Devices and methods for biochip multiplexing

ABSTRACT

The invention is directed to devices that allow for simultaneous multiple biochip analysis. In particular, the devices are configured to hold multiple cartridges comprising biochips comprising arrays such as nucleic acid arrays, and allow for high throughput analysis of samples.

This is a continuation of U.S. Ser. No. 10/412,660, filed Apr. 11, 2003,pending, which is a continuation-in-part of PCT/US01/44364, filed Nov.5, 2001 and is a continuation of U.S. Ser. No. 10/193,712, filed Jul.11, 2002, now abandoned, which is a continuation-in-part of U.S. Ser.No. 09/993,342, filed Nov. 5, 2001, pending, which is acontinuation-in-part of U.S. Ser. No. 09/904,175, filed Jul. 11, 2001,pending, which is a continuation-in-part of PCT/US01/01150, filed Jan.11, 2001 and is a continuation-in-part of U.S. Ser. No. 09/760,384,filed Jan. 11, 2001, pending, which claims the benefit under 35 U.S.C. §119(e) of U.S. Ser. No. 60/245,840, filed Nov. 3, 2000 and U.S. Ser. No.60/175,539, filed Jan. 11, 2000, all hereby expressly incorporated byreference in their entireties.

FIELD OF THE INVENTION

The invention is directed to devices that allow for simultaneousmultiple biochip analysis. In particular, the devices are configured tohold multiple cartridges comprising biochips comprising arrays such asnucleic acid arrays, and allow for high throughput analysis of samples.

BACKGROUND OF THE INVENTION

There are a number of assays and sensors for the detection of thepresence and/or concentration of specific substances in fluids andgases. Many of these rely on specific ligand/antiligand reactions as themechanism of detection. That is, pairs of substances (i.e. the bindingpairs or ligand/antiligands) are known to bind to each other, whilebinding little or not at all to other substances. This has been thefocus of a number of techniques that utilize these binding pairs for thedetection of the complexes. These generally are done by labeling onecomponent of the complex in some way, so as to make the entire complexdetectable, using, for example, radioisotopes, fluorescent and otheroptically active molecules, enzymes, etc.

Other assays rely on electronic signals for detection. Of particularinterest are biosensors. At least two types of biosensors are known;enzyme-based or metabolic biosensors and binding or bioaffinity sensors.See for example U.S. Pat. Nos. 4,713,347; 5,192,507; 4,920,047;3,873,267; and references disclosed therein. While some of these knownsensors use alternating current (AC) techniques, these techniques aregenerally limited to the detection of differences in bulk (ordielectric) impedance.

There are a variety of nucleic acid biosensors currently known. Theseinclude nucleic acid biochips based on fluorescent detection; see forexample materials developed by Affymetrix (including, but not limitedto, 5,800,992, 5,445,934, 5,744,305, and related patents and materials),Nanogen (including, but not limited to, 5,532,129, 5,605,662, 5,565,322and 5,632,957 and related patents and materials), Southern (EP 0 373 023B1) and Synteni/Incyte (WO 95/35505 and related patents and materials).Similarly, electronic detection of nucleic acids using electrodes isalso known; see for example U.S. Pat. Nos. 5,591,578; 5,824,473;5,705,348; 5,780,234 and 5,770,369; U.S. Ser. Nos. 08/813,59808/911,589; and WO 98/20162; PCT/US98/12430; PCT/US98/12082;PCT/US99/10104; PCT/US99/01705, and PCT/US99/01703 and relatedmaterials.

Integrated devices comprising biosensors and microfluidic devices areknown. For example, Jacobson and Ramsey used a single glass chip toperform an enzymatic reaction, capillary electrophoretic separation anddetection (Jacobson and Ramsey, (1996) Anal. Chem., 66: 720-723).Woolley, et al., coupled microfabricated silicon PCR reactors and glasscapillary electrophoresis chips to form an integrated DNA analysissystem (Woolley, et al., (1996) Anal. Chem., 68:4081-4086). Waters, etal., describe a single microchip device that can perform cell lysis,multiplex PCR amplification, capillary electrophoresis, and detection(Waters, et al., (1996) Anal. Chem., 70: 158-162). Burns et al.,describe a microfluidic devices that uses microfabricated fluidicchannels, heaters, temperature sensors and fluorescence detectors toanalyze nanoliter-size DNA samples (Burns, et al., (1998) Science, 282:484-487). Anderson, et al., describe an integrated genetic analysissystem consisting of passive glass chips packaged in a plastic cartridge(Anderson, et al., (1998) Solid-State Sensor and Actuator Workshop,Hilton Head Island, S.C., June 8-11, pp 7-10). Pneumatically actuatedvalves, pumps and porous hydrophobic vents are used to move fluidsthrough the system. Extracted DNA and pre-mixed reagents for PCR areintroduced through the inlet port on the cartridge. The amplicons gothrough a series of reactions inside the cartridge and the labeledtargets are detected by hybridization oligo array.

However, to date none of these methods have been used to develop anintegrated microfluidic device for the multiplex analysis of biochips.Accordingly, it is an object of the present invention to provide devicesand methods for the multiplex analysis of biochips, particularly nucleicacid biochips.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides biochip cartridges comprising one or more reaction chambers,such as a nucleic acid amplification chamber. The chambers areconfigured to include inlet and outlet ports, valves to control themovement of fluid into and out of the chamber and pumps.

In an additional aspect, the biochip cartridge comprises a detectionchamber with an array of electrodes.

In an additional aspect, the biochip cartridge comprises one or morethermal heaters.

DETAILED DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1J depict a number of different detection chamber embodiments.FIGS. 1A-1F depict alternative detection chamber geometries in which theinlet port 100 is positioned at the top of the chamber. In contrast, theoutlet port 101 can have several configurations. For example, in FIG. 1Athe outlet port is located at the top of the chamber but does not ventto the outside. In FIGS. 1B and 1D, the outlet port 101 is positioned atthe top and vents outside. In FIG. 1C, the outlet port 101 is located tothe side of the chamber. In FIG. 1F, the outlet port 101 comes off ofthe inlet port 100. In FIGS. 1A through 1E, the electrode array 103 ispositioned within a reaction chamber 102, which may be formed from agasket, a recess in the underlying printed circuit board or from thehousing 104. In FIGS. 1A and 1C, the reaction chamber is shaped like aninclined diamond; in FIG. 1B, the reaction chamber is shaped like adiamond; in FIG. 1D, the reaction chamber is circular in shape; in FIG.1E, the reaction chamber is triangular in shape; and, in FIG. 1F, thereaction chamber is shaped like a square. FIG. 1F depicts an embodimentin which reference electrodes 106 are located in the inlet and/or theoutlet channels. The reference electrodes are preferably AgCl. Thereference electrodes may be coated with AgCl before placing in thecartridge. Alternatively, a coating of AgCl may be applied to thereference electrodes while in the cartridge by applying a voltage ofsufficient strength to an Ag electrode such that the silver is oxidizedto form Ag+. FIG. 1G depicts a biochip 105 comprising a reaction chamber102 with an electrode array 103, a PCR chamber 115, a buffer chamber170, an air pump chamber or other mechanism for moving fluid 116, one ormore valves for controlling the movement of fluid 171, temperaturesensors 172, heating elements integrated into the device 173, a mixingelement 174, reference electrodes 106, inlet 100 and outlet 101 ports, amicrochannel 110, a silicon gasket 104B with a cutout for the detectionchamber 113 and a cap 130. FIG. 1H depicts the top surface of anelectronic biochip. The electrode array 103 is configured such that eachelectrode is connected via a wire lead 109 to a contact pad orinterconnect 108 on the edge of the biochip. These metal contact padscan be used to make contact between the electronic biochip reader andthe biochip using a standard computer edge card connector.Alternatively, as depicted in FIGS. 1I and 1J, the electricalconnections can be made by transversing through the board to theopposite side of the substrate. The opposite side of the substrate canbe arranged in the mirror image configuration to the front side, or itcan be arranged in an alternative fashion. FIG. 1K shows a biochip 105with connects that transverse the board making contact with a pogo pinconnector 176. The connector has an array of compliant pins 177, acircuit board housing 177, and potentially an electronic multiplexer178. The pin grid connector ultimately plugs into an instrument throughsome interface like an edge card connector via metal fingers 179. Inorder to ensure a good connection between the pogo pin connectors andthe chip, it is common to use some type of fastener.

FIG. 2 depicts the various components that can comprise a cartridge. Inthe cartridge embodiment depicted in FIG. 2A, the detection chamber 102contains an electrode array 103 connected via wires 109 to interconnects108. The array is attached to a solid surface 105 which can be made fromany number of materials as described below. In addition, registrationpins 107 can be attached to the biochip to enable the addition of othercomponents. FIG. 2B depicts a rubber gasket 104 with a cut out for thedetection chamber 113 and registration holes 112 for attaching thegasket to the biochip depicted in FIG. 2A. FIG. 2C depicts a housing114, which can be made from plastic and is attached to the biochip viaregistration holes 112. The cartridge 114, may optionally contain acutout 113A for the detection chamber 103 and a recessed microchannel110 running from the inlet port 100 to the detection chamber 103.

FIGS. 3A and 3B depict two views, top view (FIG. 3A) and an angled frontview (FIG. 3B) of the cartridge holders 129 used to hold the cartridgesduring sample loading. As shown in FIGS. 3A and 3B, the cartridge holder129, can hold several cartridges 114 with attached biochips 105. Alsoshown is a cartridge cap 130, which can be taken on and off via a“snap-in lock”.

FIGS. 3C and 3D depict different views of a cartridge with a cap. InFIG. 3C, the cap 130, is configured to include a snap-in lock 132, thatlocks into a slot 133 in the cartridge 114. In the embodiment shown inFIG. 3C, the cap has been configured to include a seal 134 for thesample introduction module 136. Preferably, the seal 134 comprises aplastic plug surrounded by cellulose or another hydrophobic materialthat allows air to pass but not liquid. The cartridge 114 has beenconfigured to include sample introduction module 136, and a PCR chamber115. The cartridge 114 is attached to a biochip comprising a reactionchamber 102, an electrode array 103, connected via wires 109 tointerconnects 108, a microchannel 110, and an outlet port 101. FIG. 3Dillustrates a side vies of the cap 130, the cartridge 114 configured tocontain a PCR chamber 115, and a chip 105.

FIG. 3E depicts a side view of several cartridge assemblies lined up asthey would appear in the cartridge holder. The cap 130, is attached tothe cartridge 114, which has been configured to comprise a sampleintroduction module 136 and a PCR chamber 115. Attached to the cartridgeis a biochip 105.

FIGS. 4A and 4B depict different views of a multiplexing device 137. InFIG. 4A, a side and top view of the multiplexing device 137 illustratesthe cartridge/station pairs 139 and a drawer 138. In FIG. 4B, themultiplexing device 137 is illustrated with the stations 141 for holdingthe cartridges and an open drawer 138.

FIG. 5 illustrates a schematic of an electronic circuit designed tomonitor the sample temperature with a thermal sensor inside thecartridge. This version of the design uses a resistive temperaturedevice composed of a copper trace. The circuitry can be used in feedbacksystem for thermal control of the cartridge temperature.

FIG. 6 illustrates the thermal control logic. This feedback mechanismemploys a Proportional Integral Derivative (PID) algorithm.

FIG. 7 illustrates the layout of a multiplexing device. The device haseight independent modules. In this particular figure, each module hassix card-edge connectors 141 and a signal processing printed circuitboard 140. Directly underneath the modules is the power supply 145.Adjacent to the power supply is a bus bar 150 for power distribution.FIG. 7 depicts the stations into which the cartridges are inserted. Acartridge 114 with a cap 130 is shown inserted into an card edgeconnector 141.

FIG. 8 is a block diagram for the software application used to controlthe multiplexing device.

FIGS. 9A-N depict a variety of different valves that may be used in thepresent invention. FIG. 9A depicts a duck bill valve 144 within amicrochannel 110 that can be used to control the flow of liquid in onedirection, but not the other. FIG. 9B depicts a cantilever valve 146. Inthis embodiment, voltage, applied via electrodes 147 is used to open andclose the cantilever valve 146. FIG. 9C depicts a plunger type valvemechanism. In this embodiment, a plunger valve 148 can be opened andclosed via the use of a shape memory wire 149. FIGS. 9D and 9E depictrotary valves. In the embodiment shown in FIG. 9D, an external forcemust be applied to rotate the rotary valve 151. In FIG. 9E, a shapememory wire 149 is used to rotate the rotary valve 151. FIG. 9F depictsa thermally actuated valve that comprises a portion of the microchannel110 with a flexible membrane 152 that can be filled with liquid or air153 for use in conjunction with a heater 154. FIG. 9G depicts a genericpump design in which a chamber 156 through which air and/or liquid canflow is attached to an inlet port 100 with a valve, such as a cantilevervalve 146, to control movement into the chamber and an outlet port 101with a valve 146A, to control movement out of the chamber. An externaldevice 181, such as a PZT, can be used to compress chamber 156.Alternatively, a heater 182, can be actuated expanding the volume of thegas or liquid in chamber 156. FIG. 9H depicts an example of athermopneumatically actuated PDMS microvalve comprising a heater 182, avertrel chamber 190, and a PDMS membrane 191. FIG. 9I depicts themulti-layer PDMS (193) microfabrication used to construct the valve.FIG. 9J depicts a “close-open” paraffin valve comprising a wax valve195, a thermal zone 105, and a microchannel 110. FIG. 9K depicts a“open-close-open” paraffin valve comprising a wax valve 195, a thermalzone 105, an air pump 196 and a microchannel 110. FIG. 9L depicts a “T”paraffin valve. FIG. 9M depicts a “toggle” paraffin valve. FIG. 9Ndepicts a Pluronics valve comprising a chamber 197 to hold the Pluronicsgel 198.

FIG. 10A is a side view of a biochip 105 depicting an embodiment inwhich a thermal heater is incorporated into the chip. In FIG. 10 A, theresistive heaters 157 are overlaid with a layer of copper 158. Thecopper layer is overlaid with printed circuit board 125, which iscovered with solder mask 159.

FIG. 10B illustrates one means of creating thermal zones in a biochip105. In the embodiment shown in FIG. 10B, successive thermal zones 161,162, 163 comprising several rows of resistive heaters 157 are overlaidwith a serpentine microchannel 164. Discrete temperature zones aremaintained by controlling the minimum separation distance betweenresistive heater 161, 162, and 163 as well as varying the thermalproperties of the separating materials. the device illustrated in FIG.10B can be used in conjunction with a pumping device to transport afluid between temperature zones and perform biological reactions thatrequire heat cycling like PCR.

FIG. 10C depicts a biochip analogous to FIG. 10B, but made out ofceramic 165 with imbedded heaters 166 and corresponding edge connections108. The temperature difference between the thermal zones is maintainedby creating air pocket cut-outs 167 that have a lower thermallyconductivity than the surrounding ceramic.

FIG. 11 depicts a bar code reader 142 reading a bar code 143.

FIG. 12 describes a bar code usage scenario.

FIG. 13 highlights the benefits of using a bar code in combination withthe devices of the present invention.

FIGS. 14 A and B depict a preferred embodiment of a biochip 105 withregistration holes 112 for the attachment of a cartridge 114. In FIG.14A, the top surface of a biochip 105 is depicted showing theregistration holes 112, an electrode array 103 and interconnects 108. InFIG. 14B, a cartridge 114 is shown overlaying the biochip 105illustrated in FIG. 14A. In the embodiment shown, the cartridge 114includes registration pins 107 for attaching the cartridge to thebiochip. Preferably, the registration pins are plastic. Also shown areinlet 100 and outlet 101 ports, microchannels 110, a PCR chamber 115, areaction chamber 102 and a sample introduction chamber 136. The capgasket 134 is depicted as an insert within the sample introductionchamber 136.

FIG. 15A depicts sample loading using a pipet tip 144 into a cartridge114 inserted into a station 141 of a multiplexing device 137.

FIGS. 15B and C depict an alternative embodiment for attaching acartridge to a biochip. In FIG. 15A, the biochip 105 is designed to havealignment slots 118. In FIG. 15C, the cartridge is configured to haveregistration pins 107 that fit into the registration holes on the sidesof the biochip. Also depicted in FIG. 15C is the use of a pipet tip 144for loading a sample into a sample introduction chamber 136.

FIG. 16 depicts a sine wave and its corresponding vector notation.

FIG. 17 depicts the visualization of the sine wave shown in FIG. 16using vector notation. The two values can be R and θ, but as shown inthe FIG. 17 they can also be an (X,Y) pair separated by one quarter ofan oscillation, i.e. by 90°.

FIGS. 18 and 19 are examples of R and θ traces for the fourth harmonicACE voltammetry.

FIGS. 20 and 21 depict that the R space signal distorts as the signalshrinks relative to the size of the background.

FIGS. 22 through 25 illustrate how the use of Cartesian coordinatessimplifies the dependence of D's parameters on those of S and B. Thissimplicity is exhibited when graphing the same examples shownpreviously, but now as (X,Y), as depicted in FIGS. 22 and 23 (mediumsized signal), and FIGS. 24 and 25 (smaller signal).

FIG. 26 depicts an AC voltage-4 trace that has a large signal relativeto the background. FIG. 26 is obtained 1 f, in a two-dimensional graph,the tip of the data vector as a function of voltage (one point isplotted every 10 mV) is plotted.

FIGS. 27 and 28 depict the result when a frame of reference is chosensuch that the X and Y axes straddles the signal. In this case, thesignal contributes strongly to both X and Y.

FIGS. 29 and 30 depicts the signal that is observed when an axis pairthat is roughly parallel and perpendicular to the signal (rotated 45

with respect to the axes drawn in FIG. 26) is chosen. In this case, verylittle of the signal contributes to the perpendicular vector.

FIGS. 31 through 35 illustrate the vectoral sum method. For signalrecognition based on the AC voltage 4 trace model, a new pair of axesneed to be chosen to straddle any existing electrochemical signal. Inorder to choose such axes, a way is needed to measure the signal'sdirection. One such way is using a vectoral sum. Consider the groupingof three points shown in FIG. 31. If we consider these points asvectors, we can add them by summing their coordinates. This summation ofthe vectors provides a reasonable angle for the best line through thedata that passes through (0,0). This angle is called the “optimalphase.” For our example, the summation is drawn in FIG. 32. FIG. 33shows how the three sample data points cluster around the line. Anadvantage to this method is that the results are weighted by the lengthof the vectors of the original data points. For example, if we add asmall data point to the sample grouping, the results are shown in FIGS.34 and 35.

FIGS. 36 through 41 illustrate the complications that must be consideredwhen using the vectoral sum to calculate the optimal phase for fitting asignal. For example, if the electrochemical signal is shaped such thatportions of it cancel each other out when completing the calculationdescribed above, the first one half of the data must be rotated 180

. Taking the data shown in FIG. 26, we calculate the optimal phase usingthe data as shown in FIG. 36. The resulting line is overlaid on theoriginal data, at FIG. 37. The angle of the line drawn in FIGS. 36 and37 (101

) is what was used to choose the X and Y axes (at ±45

) for this file. However, if the signal is oriented differently relativeto the dividing line between rotated and unrotated segments, the statedmanipulation may not yield the proper angle. For example, if I take theabove signal and rotate it 101 degrees clockwise, its optimal phaseshould be 0

. However, the calculated value actually ends up as −48

as shown in FIG. 38. To prevent this, a rotation boundary that is moreperpendicular to the signal than it is parallel is chosen. Taking thevectoral sum of the absolute value of the coordinates of a signal that'scloser to 90, the resulting angle will be greater than 45

. Thus, for the above case we find an angle of 10 degrees (see FIG. 39),less than 45, and conclude that the signal is more along 0 degrees.Therefore, we rotate the half of the signal from the far side of the 90degree axis (see FIG. 40). Calculating the vectoral sum now yields areasonable value for the optimal phase: 1

, similar to the expected 0

. When the scan is examined in two dimensions, we can see that the phaseof the entire scan is mostly along 120

(FIG. 41).

FIGS. 42 through 46 illustrates the results obtained if the rapidcalculations necessary to fit polynomials to the entire scan (one eachalong the 0 and 90

axes) are performed. For example, the background is approximated asshown in FIGS. 42 and 43. The approximation to the background can besubtracted, converting the scan into something that is much more purelysignal, as shown in FIGS. 44 and 45. FIG. 46 depicts this as a twodimensional plot, from which an optimal phase of approximately 70

can be calculated.

FIGS. 47 through 52 illustrate how behaviors not modeled are detected.To reduce total processing time, the first thing to do is to check if ascan has any gross deviations from the model that would make fitting itmeaningless. One such feature encountered in AC voltammetry (fourthharmonic) has been the sharp peak caused by the stripping of a metalliccontaminant. FIG. 47 shows an example of one displayed in R-space. In Xand Y (at ±45

from the optimal phase), the sharp spike feature remains clear, as shownin FIGS. 48 and 49.

The symmetry of this feature distinguishes it from our normal signal.One method of monitoring this symmetry is to separate out an approximatebackground and compare the distribution of points above the baselinewith the distribution below. For example, if we subtract a polynomialfrom the Y trace above, we get the results shown in FIGS. 50 and 51. Ifwe now examine the distribution of data above and below the approximatedbackground, the presence of the spike causes a larger range of values toexist below the background line than above it, as shown in FIG. 52.

FIGS. 53 through 58 illustrate the initial guess process required as astarting point for iterative fitting procedures. To guess parameters ofsignal position and signal height, the known AC voltage-4 symmetry isused combined with knowledge of the characteristic width. Since theaverage separation between the two larger center lobes is known, thesignal and shift are duplicated, the two copies in opposite directionsfor half of that separation. Subtracting one from the other, the centerlobes interfere constructively. The absolute value of this resultingwave provides a good estimation of the height and position of thesignal. This process is shown in FIGS. 53, 54 and 55 for a signal 11.9tall at a position of 0.20 with a center lobe separation of 0.072. Thetrace in FIG. 55 has its largest value, 23.25, at a position of 0.20.The position matches well with the true data value. (Both are 0.20.)

In FIG. 56, the same signal is considered, but this time with an unusualpeak off to one side that's slightly taller than the signal itself. In asimple maxima/minima search, this would be likely to interfere with theinitial guess. However, using the initial guess, the signal remains 11.6tall at a position of 0.20, as shown in FIG. 57. FIG. 58 is the overlayof a real data trace and the corresponding initial guess.

FIGS. 59 through 61 depict that for systems that are less well-behaved,the boundary conditions may be enforced during the fitting procedure.This can be done using various equations described below. In FIG. 59 theshape when the added term has 2n=16 is compared with the shape when2n=2. In the case where 2n=16, a values within ±7 of the expected areall equally acceptable, with little added penalty. However, with 2n=2,there's an increasingly harsh penalty the further a moves from theexpected value. Sharper constraints a result in the shapes depicted inFIG. 60. More complicated shapes may be used, as shown in FIG. 61.

FIG. 62 depicts the results when the fit is not reliable because thedifference between the fit and date is too large.

FIGS. 63 through 65 depict the results obtained using procedures torefit scans having no observable signals.

FIGS. 66 and 67 depict the results obtained using the proceduresdescribed herein. FIG. 66 depicts the original data. FIG. 67 depicts thedata with the background subtracted.

FIG. 68 depicts the effect of various plasma treatments on the surfacedensity of SAMs comprising capture probes.

FIG. 69 depicts the spectra of contaminants on a gold surface aftervarious plasma treatment procedures. The major contaminant peaks ofcarbon and oxygen are marked out with gold peaks for the oxygen plasmatreatment alone, or for the oxygen plasma followed by hydrogen plasmatreatment.

FIG. 70A-F depict the effect of different mixing techniques onhybridization kinetics in an eSensor™ chamber. FIG. 70A depicts effectof chip orientation (i.e., diffusion based kinetics) on hybridizationkinetics and increased volume/z-dimension. Increased volume was obtainedby increasing the thickness of the chamber using one (single tape), two(double tape) or three (triple tape) layers of tape. Chips wereincubated either horizontally (H) or vertically (V). FIG. 70B comparesdiffusion based kinetics using chips oriented either vertically (vdiff)or horizontally (hdiff) to mixing using a recirculation pump (vpump).FIG. 70C compares diffusion based kinetics/vertical orientation (vdiff)to bubble assisted PZT mixing using either a square wave (vpztsquare) ora sine wave excitation (vpztsine) waveform. FIG. 70D compares diffusionbased kinetics/vertical orientation (vdiff) to thermal gradient basedmixing (TG). FIG. 70E compares diffusion based kinetics/verticalorientation to diffusion based kinetics in a biochannel/verticalorientation to diffusion based kinetics in a biochannel/horizontalorientation to bubble mixing in a biochannel/horizontal orientation.FIG. 70F depicts acoustic based mixing (treat-H) to diffusion basedkinetics using either vertical (ctrl-V) or horizontal (ctrl-H) chiporientation.

In FIG. 71, there is illustrated a schematic block diagram of anexemplary signal processing approach. A digital to analog converter(DAC) receives a digital signal from a signal source (such as signalgenerating circuitry on the signal processing printed circuit board orreceived from a connected personal computer) and converts that signalinto an analog signal which is received by filter. The characteristicsof filter may be modified to provide frequency low-pass, high-pass, orsingle or multiple band-pass characteristics according to tailored thesignal applied to the electrodes of the E-Chem Cell. In this embodiment,the filtered signal is passed through resistor R9 (110 Kohm) beforepassing through a first auxiliary amplifier (AUX AMP). To reduce signalcomplexity and cost, the signal is desirably multiplexed throughmultiplexer (MUX) and distributed to a plurality of auxiliary electrodeson the E-Chem cell cartridge.

A set of reference electrodes is also disposed within the E-Chem Cellcartridge, the outputs of which are coupled to through a secondmultiplexer (MUX) and reference amplifier (REF AMP) and resistor R13(110 Kohm) back to the input of first auxiliary amplifier.

Finally, a set of active electrodes (36 active electrodes in thisembodiment) are coupled via printed circuit board traces to a thirdmutiplexer. The output of this active electrode multiplexer is amplifiedby an input signal amplifier (INPUT AMP), and after further optionalsignal conditioning (such as filtering, gain control and/or selection)is processed through a buffer amplifier (BUFFER AMP) and converted fromanalog to digital (ADC) form, so that it may be communicated, processed,analyzed, stored or the like in digital form.

In FIG. 72 there is illustrated an embodiment of a Thermal Control BlockDiagram. A microprocessor communicates with an external signal source orsink via a serial communication channel or link.

Advantageously, microprocessor generates a control signal to provide anindication of an ON, OFF, or FLASH status to an LED logic circuit whichis coupled with and causes LED Drivers to send signals to each of sixslots causing each of the slots red or green lights to be on, off, orflash. Microprocessor also generates a signal to DAC. This signal isamplified to power a heat sink blower to control the temperature of theheat sink. A heat sink temperature sensor is associated with the heatsink and this sensor generates a temperature signal which is fed back tothe microprocessor in feedback manner to control operation ornon-operation of the heat sink blower motor.

The microprocessor also generates a plurality of signals which arereceived by a plurality of DAC and driver amplifiers to a Peltierthermal block for each slot. A temperature sensor is also associatedwith each Peltier thermal block to provide a sensed temperatureindication back to the microprocessor for controlling the Peltierthermal block drive signal in feedback manner.

In FIG. 73 there is illustrated an exemplary layout for a signalprocessing printed circuit board. Each board includes an edge connectorfor coupling with a communication bus, motherboard, or otherinterconnect as are known in the art. Each board in this particularembodiment further includes pad selector circuitry, memory, aCPU/lock-in amplifier, buffers, serial communication circuitry, waveformsignal generators, analog-to-digital converter (ADC), filters or filtercircuits, master gain circuit, current-to-voltage converter, powerregulators, and chip selector (ref/mux).

FIG. 74A depicts an inductive antenna. FIG. 74B depicts a capacitiveantenna.

DETAILED DESCRIPTION

The present invention is directed to devices designed to receive andanalyze a plurality of biochips, each comprising an array of biologicalmoieties, such as nucleic acids or proteins, to allow high throughputanalysis and detection of target analytes in samples. Thus for example anumber of samples (particularly patient samples) can be simultaneouslyanalyzed, or multiple assays can be run on a single sample. The devicescomprise a number of cartridge stations that are configured to receivethe biochips, with different types of biochips allowing different typesof components. The stations can include a wide variety of differentcomponents, including thermocontrollers, signaling systems, sensors forleak detection, alphanumeric displays, and detectors. Preferredembodiments include the use of biochips comprising electrodes that relyon electrochemical detection, and thus the devices and/or stations cancomprise device boards and processors.

The biochip cartridges include substrates comprising the arrays ofbiomolecules, and can be configured in a variety of ways. For example,the chips can include reaction chambers with inlet and outlet ports forthe introduction and removal of reagents. In addition, the cartridgescan include caps or lids that have microfluidic components, such thatthe sample can be introduced, reagents added, reactions done, and thenthe sample is added to the reaction chamber comprising the array fordetection.

Accordingly, the present invention provides compositions and methods fordetecting the presence or absence of target analytes in samples. As willbe appreciated by those in the art, the sample solution may comprise anynumber of things, including, but not limited to, bodily fluids(including, but not limited to, blood, urine, serum, lymph, saliva, analand vaginal secretions, perspiration and semen, of virtually anyorganism, with mammalian samples being preferred and human samples beingparticularly preferred); environmental samples (including, but notlimited to, air, agricultural, water and soil samples); biologicalwarfare agent samples; research samples (i.e. in the case of nucleicacids, the sample may be the products of an amplification reaction,including both target and signal amplification as is generally describedin PCT/US99/01705, such as PCR amplification reaction); purifiedsamples, such as purified genomic DNA, RNA, proteins, etc.; raw samples(bacteria, virus, genomic DNA, etc.); as will be appreciated by those inthe art, virtually any experimental manipulation may have been done onthe sample.

The methods are directed to the detection of target analytes. By “targetanalyte” or “analyte” or grammatical equivalents herein is meant anymolecule or compound to be detected and that can bind to a bindingspecies, defined below. Suitable analytes include, but are not limitedto, small chemical molecules such as environmental or clinical chemicalor pollutant or biomolecule, including, but not limited to, pesticides,insecticides, toxins, therapeutic and abused drugs, hormones,antibiotics, antibodies, organic materials, etc. Suitable biomoleculesinclude, but are not limited to, proteins (including enzymes,immunoglobulins and glycoproteins), nucleic acids, lipids, lectins,carbohydrates, hormones, whole cells (including procaryotic (such aspathogenic bacteria) and eucaryotic cells, including mammalian tumorcells), viruses, spores, etc. Particularly preferred analytes areproteins including enzymes; drugs, cells; antibodies; antigens; cellularmembrane antigens and receptors (neural, hormonal, nutrient, and cellsurface receptors) or their ligands.

In a preferred embodiment, the target analyte is a protein. As will beappreciated by those in the art, there are a large number of possibleproteinaceous target analytes that may be detected using the presentinvention. By “proteins” or grammatical equivalents herein is meantproteins, oligopeptides and peptides, derivatives and analogs, includingproteins containing non-naturally occurring amino acids and amino acidanalogs, and peptidomimetic structures. The side chains may be in eitherthe (R) or the (S) configuration. In a preferred embodiment, the aminoacids are in the (S) or L-configuration. As discussed below, when theprotein is used as a binding ligand, it may be desirable to utilizeprotein analogs to retard degradation by sample contaminants.

Suitable protein target analytes include, but are not limited to, (1)immunoglobulins, particularly IgEs, IgGs and IgMs, and particularlytherapeutically or diagnostically relevant antibodies, including but notlimited to, for example, antibodies to human albumin, apolipoproteins(including apolipoprotein E), human chorionic gonadotropin, cortisol,α-fetoprotein, thyroxin, thyroid stimulating hormone (TSH),antithrombin, antibodies to pharmaceuticals (including antieptilepticdrugs (phenyloin, primidone, carbariezepin, ethosuximide, valproic acid,and phenobarbitol), cardioactive drugs (digoxin, lidocaine,procainamide, and disopyramide), bronchodilators (theophylline),antibiotics (chloramphenicol, sulfonamides), antidepressants,immunosuppresants, abused drugs (amphetamine, methamphetamine,cannabinoids, cocaine and opiates) and antibodies to any number ofviruses or bacteria outlined below.

As will be appreciated by those in the art, a large number of analytesmay be detected using the present methods; basically, any target analytefor which a binding ligand, described below, may be made may be detectedusing the methods of the invention.

In a preferred embodiment, the target analytes are nucleic acids. By“nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al.,Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993);Carlsson et al., Nature 380:207 (1996), all of which are incorporated byreference). Other analog nucleic acids include those with bicyclicstructures including locked nucleic acids, Koshkin et al., J. Am. Chem.Soc. 120:13252-3 (1998); positive backbones (Denpcy et al., Proc. Natl.Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos.5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi etal., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J.Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside &Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett.4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994);Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, “Carbohydrate Modifications inAntisense Research”, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acidscontaining one or more carbocyclic sugars are also included within thedefinition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995)pp169-176). Several nucleic acid analogs are described in Rawls, C & ENews Jun. 2, 1997 page 35. All of these references are hereby expresslyincorporated by reference. These modifications of the ribose-phosphatebackbone may be done to facilitate the addition of ETMs, or to increasethe stability and half-life of such molecules in physiologicalenvironments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made; for example,at the site of conductive oligomer or ETM attachment, an analogstructure may be used. Alternatively, mixtures of different nucleic acidanalogs, and mixtures of naturally occurring nucleic acids and analogsmay be made.

Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids. Thisresults in two advantages. First, the PNA backbone exhibits improvedhybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4

C drop in Tm for an internal mismatch. With the non-ionic PNA backbone,the drop is closer to 7-9

C. Similarly, due to their non-ionic nature, hybridization of the basesattached to these backbones is relatively insensitive to saltconcentration.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions, of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. A preferred embodimentutilizes isocytosine and isoguanine in nucleic acids designed to becomplementary to other probes, rather than target sequences, as thisreduces non-specific hybridization, as is generally described in U.S.Pat. No. 5,681,702. As used herein, the term “nucleoside” includesnucleotides as well as nucleoside and nucleotide analogs, and modifiednucleosides such as amino modified nucleosides. In addition,“nucleoside” includes non-naturally occurring analog structures. Thusfor example the individual units of a peptide nucleic acid, eachcontaining a base, are referred to herein as a nucleoside.

Thus, in a preferred embodiment, the target analyte is a targetsequence. The term “target sequence” or “target nucleic acid” orgrammatical equivalents herein means a nucleic acid sequence on a singlestrand of nucleic acid. The target sequence may be a portion of a gene,a regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA,or others. As is outlined herein, the target sequence may be a targetsequence from a sample, or a secondary target such as a product of anamplification reaction, etc. It may be any length, with theunderstanding that longer sequences are more specific. As will beappreciated by those in the art, the complementary target sequence maytake many forms. For example, it may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others. As is outlined morefully below, probes are made to hybridize to target sequences todetermine the presence or absence of the target sequence in a sample.Generally speaking, this term will be understood by those skilled in theart. The target sequence may also be comprised of different targetdomains; for example, a first target domain of the sample targetsequence may hybridize to a capture probe or a portion of captureextender probe, a second target domain may hybridize to a portion of anamplifier probe, a label probe, or a different capture or captureextender probe, etc. The target domains may be adjacent or separated asindicated. Unless specified, the terms “first” and “second” are notmeant to confer an orientation of the sequences with respect to the5′-3′ orientation of the target sequence. For example, assuming a 5′-3′orientation of the complementary target sequence, the first targetdomain may be located either 5′ to the second domain, or 3′ to thesecond domain.

Suitable target analytes include biomolecules associated with: (1)viruses, including but not limited to, orthomyxoviruses, (e.g. influenzavirus), paramyxoviruses (e.g. respiratory syncytial virus, mumps virus,measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses,togaviruses (e.g. rubella virus), parvoviruses, poxviruses (e.g. variolavirus, vaccinia virus), enteroviruses (e.g. poliovirus, coxsackievirus),hepatitis viruses (including A, B and C), herpesviruses (e.g. Herpessimplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barrvirus), rotaviruses, Norwalk viruses, hantavirus, arenavirus,rhabdovirus (e.g. rabies virus), retroviruses (including HIV, HTLV-I and-II), papovaviruses (e.g. papillomavirus), polyomaviruses, andpicornaviruses, and the like; and (2) bacteria, including but notlimited to, a wide variety of pathogenic and non-pathogenic prokaryotesof interest including Bacillus; Vibrio, e.g. V. cholerae; Escherichia,e.g. Enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella,e.g. S. typhi; Mycobacterium e.g. M. tuberculosis, M. leprae;Clostridium, e.g. C. botulinum, C. tetani, C. difficile, C. perfringens;Cornyebacterium, e.g. C. diphtheriae; Streptococcus, S. pyogenes, S.pneumoniae; Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H.influenzae; Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia,e.g. G. lamblia Y. pestis, Pseudomonas, e.g. P. aeruginosa, P. putida;Chlamydia, e.g. C. trachomatis; Bordetella, e.g. B. pertussis;Treponema, e.g. T. palladium; and the like.

Other suitable target analytes include, but are not limited to, (1)enzymes (and other proteins), including but not limited to, enzymes usedas indicators of or treatment for heart disease, including creatinekinase, lactate dehydrogenase, aspartate amino transferase, troponin T,myoglobin, fibrinogen, cholesterol, triglycerides, thrombin, tissueplasminogen activator (tPA); pancreatic disease indicators includingamylase, lipase, chymotrypsin and trypsin; liver function enzymes andproteins including cholinesterase, bilirubin, and alkaline phosphotase;aldolase, prostatic acid phosphatase, terminal deoxynucleotidyltransferase, and bacterial and viral enzymes such as HIV protease; (2)hormones and cytokines (many of which serve as ligands for cellularreceptors) such as erythropoietin (EPO), thrombopoietin (TPO), theinterleukins (including IL-1 through IL-17), insulin, insulin-likegrowth factors (including IGF-1 and -2), epidermal growth factor (EGF),transforming growth factors (including TGF-α and TGF-β), human growthhormone, transferrin, epidermal growth factor (EGF), low densitylipoprotein, high density lipoprotein, leptin, VEGF, PDGF, ciliaryneurotrophic factor, prolactin, adrenocorticotropic hormone (ACTH),calcitonin, human chorionic gonadotropin, cotrisol, estradiol, folliclestimulating hormone (FSH), thyroid-stimulating hormone (TSH), leutinzinghormone (LH), progeterone and testosterone; and (3) other proteins(including α-fetoprotein, carcinoembryonic antigen CEA, cancer markers,etc.).

Suitable target analytes include carbohydrates, including but notlimited to, markers for breast cancer (CA15-3, CA 549, CA 27.29),mucin-like carcinoma associated antigen (MCA), ovarian cancer (CA125),pancreatic cancer (DE-PAN-2), prostate cancer (PSA), CEA, and colorectaland pancreatic cancer (CA 19, CA 50, CA242).

Other suitable target analytes include metal ions, particularly heavyand/or toxic metals, including but not limited to, aluminum, arsenic,cadmium, selenium, cobalt, copper, chromium, lead, silver and nickel.

In a preferred embodiment, the methods of the invention are used todetect pathogens such as bacteria. In this embodiment, preferred targetsequences include rRNA, as is generally described in U.S. Pat. Nos.4,851,330; 5,288,611; 5,723,597; 6,641,632; 5,738,987; 5,830,654;5,763,163; 5,738,989; 5,738,988; 5,723,597; 5,714,324; 5,582,975;5,747,252; 5,567,587; 5,558,990; 5,622,827; 5,514,551; 5,501,951;5,656,427; 5,352,579; 5,683,870; 5,374,718; 5,292,874; 5,780,219;5,030,557; and 5,541,308, all of which are expressly incorporated byreference.

As will be appreciated by those in the art, a large number of analytesmay be detected using the present methods; basically, any target analytefor which a binding ligand, described below, may be made may be detectedusing the methods of the invention. While many of the techniquesdescribed below exemplify nucleic acids as the target analyte, those ofskill in the art will recognize that other target analytes can bedetected using the same systems.

If required, the target analyte is prepared using known techniques. Forexample, the sample may be treated to lyse the cells, using known lysisbuffers, electroporation, etc., with purification and/or amplificationas needed, as will be appreciated by those in the art. When the targetanalyte is a nucleic acid, the target sequence may be amplified asrequired; suitable amplification techniques are outlined in PCTUS99/01705, hereby expressly incorporated by reference. In addition,techniques to increase the amount or rate of hybridization can also beused; see for example WO 99/67425 and U.S. Ser. Nos. 09/440,371 and60/171,981, all of which are hereby incorporated by reference.

The samples comprising the target analytes can be added to cartridgescomprising the biochips as is outlined in greater detail below. By“cartridge” herein is meant a casing or housing for the biochip. Asoutlined herein, and as will be appreciated by those in the art, thecartridge can take on a number of configurations and can be made of avariety of materials. Suitable materials include, but are not limitedto, fiberglass, teflon, ceramics, glass, silicon, mica, plastic(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polycarbonate,polyurethanes, Teflon™, and derivatives thereof, etc.), etc.Particularly preferred cartridge materials are plastic (includingpolycarbonate and polyproplylene) and glass.

As will be appreciated by those in the art, the cartridge can comprise anumber of components, including reaction chambers, inlet and outletports, heating elements including thermoelectric components, RFantennae, electromagnetic components, memory chips, sealing componentssuch as gaskets, electronic components including interconnects,multiplexers, processors, etc.

In a preferred embodiment, the cartridge comprises a reaction chamber.Generally, the reaction chamber comprises a space or volume that allowsthe contacting of the sample to the biochip array. The volume of thereaction chamber can vary depending on the size of the array and theassay being done. In general, reaction chamber ranges from 1 mL to about1 mL, with from about 1 to about 250 μl being preferred and from about10 to about 100 μl being especially preferred. In some embodiments, toavoid the introduction of air bubbles into the reaction chamber (whichcan be disruptive to detection), the reaction chamber is less than thesize of the sample to be introduced, to allow a slight overflow and thusensure that the reaction chamber contains little or no air.

In a preferred embodiment, the biochip cartridge can be configured toinclude additional chambers that can used for any number of differentreactions, such as sample preparation, cell lysis, rare targetcapture/concentration, sample clean-up, nucleic acid amplification,including PCR, post-amplification clean-up, sample concentration,reagent storage, mixing baffles/devices, etc. In other embodiments, thereaction chamber may be configured for other types of reactions asgenerally described below.

In a preferred embodiment, the biochip cartridge reaction chamber isconfigured to include at least one nucleic acid amplification chamber.However, multiple amplification chambers may be used. That is, acartridge may comprise from about 1 to about 10 or more chambers, with2, 3, 4, 5, 6, 7, 8 or 9 also being preferred.

In a preferred embodiment, the biochip cartridge reaction chamber isconfigured to include at least one PCR chamber. However, multiple PCRchambers may be used. That is, a cartridge may comprise from about 1 toabout 10 or more chambers, with 2, 3, 4, 5, 6, 7, 8 or 9 also beingpreferred.

In a preferred embodiment, the chamber of the cartridge should be madefrom biocompatible materials. In particular, materials that provide asurface that retards the non-specific binding of biomolecules, e.g. a“non sticky” surface, are preferred. For example, when the reactionchamber is used for PCR or amplification reactions a “non sticky”surface prevents enzymatic components of the reaction mixture fromsticking to the surface and being unavailable in the reaction. Inaddition, the biocompatible properties of the chamber may be improved byminimizing the surface area.

Biocompatible materials include, but are not limited to, plastic(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyimide,polycarbonate, polyurethanes, Teflon™, and derivatives thereof, etc.)Other configurations include combinations of plastic and printed circuitboard (PCB; defined below). For example at least one side of the chamberis printed circuit board, while one or more sides of the chamber aremade from plastic. In a preferred embodiment, three sides of the chamberare made from plastic and one side is made from printed circuit board.In addition, the chambers, channels, valves, pumps, etc. of the systemsdescribed herein may be coated with a variety of materials to reducenon-specific binding. These include proteins such as caseins andalbumins (bovine serum albumin, human serum albumin, etc.), parylene,other polymers, etc.

The reaction chamber of the cartridge comprises an inlet port for theintroduction of the sample to be analyzed. Depending on the reactionbeing run, multiple inlet ports may be used, that may feed from avariety of storage chambers or from the outside of the chamber. Theinlet port may optionally comprise a seal to prevent or reduce theevaporation of the sample or reagents from the reaction chamber. In apreferred embodiment (as depicted in FIGS. 3C and 14B), the sealcomprises a gasket, or valve through which a pipette or syringe can bepushed. The gasket or valve can be rubber or silicone or other suitablematerials, such as materials containing cellulose.

The reaction chamber can be configured in a variety of ways. In apreferred embodiment, the reaction chamber is configured to minimize theintroduction or retention of air bubbles or other sample impurities.Thus for example, as depicted in FIG. 1, assuming that the cartridge isheld in an upright angle, the inlet port allows the flow of fluid sampleinto the “bottom” of the reaction chamber, to allow the escape of air orfluid through the “top” of the reaction chamber, for example through anoutlet port. Thus the fluid sample flows up into the reaction chamberand contacts the array. Thus, in a preferred embodiment, the reactionchamber further comprises an outlet port to allow air or excess sampleto exit the reaction chamber. In some embodiments, the outlet port ventsto either a waste storage well, as is further described below, to anexternal surface of the chip or cartridge, or, in a preferredembodiment, back into the inlet port. Thus for example a preferredembodiment utilizes a system wherein the exit port vents to the inletport, preferably above the point of loading. For example, when a pipetteis used to load the cartridge, the tip of the pipette extends below theexit port, such that air from the exit port is not introduced into thereaction chamber. In addition, the materials of the cartridge housingand biochip can be chosen to be similar in hydrophobicity orhydrophilicity, to avoid the creation of air bubbles.

In a preferred embodiment, an anti-siphon vent is used to prevent liquidfrom being sucked back into a chamber as a result of the negativepressure generated when an air pump heater is turned off. For example, aanti-siphon vent comprising a paraffin valve and an open port can beconstructed between the reaction chamber and an air pump.

In addition, in a preferred embodiment, the reaction chamber/inletand/or outlet ports optionally include the use of valves. For example, asemi-permeable membrane or filter may be used, that preferentiallyallows the escape of gas but retains the sample fluid in the chamber.For example, porous teflons such as Gortex™ allow air but not fluids topenetrate.

In a preferred embodiment, a reaction chamber in the biochip cartridge(such as a PCR chamber) has one or more valves controlling the flow offluids into and out of the chamber. The number of valves in thecartridge depends on the number of channels and chambers. Alternatively,the biochip cartridge is designed to include one or more loading portsor valves that can be closed off or sealed after the sample is loaded.It is also possible to have multiple loading ports into a singlechamber; for example, a first port is used to load sample and a secondport is used to add reagents. In these embodiments, the biochipcartridge may have a vent. The vent can be configured in a variety ofways. In some embodiments, as generally depicted in the FIGS. 1A-1G, thevent can be a separate port, optionally with a valve, that leads out ofthe reaction chamber. Alternatively, the vent may be a loop structurethat vents liquid and/or air back into the inlet port, as generallydepicted in FIG. 1F.

As will be appreciated by those in the art, a variety of differentvalves may be used. Microvalves can be categorized into two major types:passive microvalves (without actuation) and active microvalves (with anactuation). Generally, active microvalves couple a flexible diaphragm toa thermopneumatic, piezoelectric, electrostatic, electromagnetic,bimetallic actuator.

Valves can be multi cycle or single cycle valves. By “multicycle” valvesis meant that the valve can be opened and closed more than once. By“single cycle valves” or “burst valves” or “one time valves” herein ismeant a valve that is closed and then opened or opened and then closedbut lacks a mechanism for restoring the valve to its original position.Valves may also be check valves, which allow fluid flow in only onedirection, or bi-directional valves.

In a preferred embodiment, check valves are used to prevent fluid fromgoing in and out of the reaction chamber during reactions. Generallycheck valves are used when in embodiments in which it is desirable tohave fluids and/or air flow in one direction, but not the other. Forexample, when the chamber is filled and thus compressed, air and liquidflow out. Conversely, valves can be used to empty the chamber as well.Types of check valves that can be used include, but are not limited to,duck bill valves (Vernay, www.vernay.com), cantilevers, bubble valves,etc.

In a preferred embodiment, the valve is a duck bill valve as generallydepicted in FIG. 9A. These valves are “one way” valves, in that fluidcan flow through in one direction but not the other.

In a preferred embodiment, the valve is a cantilever valve. As will beappreciated by those in the art, there are a variety of different typesof cantilever valves known in the art. Cantilever valves can also beconfigured for use in pumping systems as described below. In a preferredembodiment, a cantilever valve comprising a metal is used. In thisembodiment, the application of a voltage can either open or close avalve. See FIG. 9B.

In a preferred embodiment, a heat pump is incorporated into the systemfor opening and closing the cantilever valve. In this embodiment, thecheck valves are made out of metals such as gold and copper such thatthe check valve functions as a cantilever when heat is applied. In otherembodiments, an actuating force is not used to pull down the valve,rather they have a restraining force that prevents them from going inthe other direction.

Similarly, a thermally actuated” valve that comprises a portion of themicrochannel with a flexible membrane filled with air or liquid can beused in conjunction with a heater. The application of heat causes thefluid to expand, blocking the channel. See FIG. 9F.

In a preferred embodiment, thermopneumatically actuated valves made offlexible membrane material may be used to control flow into and out ofthe chamber(s). Suitable flexible materials includepolydimethylsiloxane, polymer sheets, silicone, rubber, etc.

In a preferred embodiment, a thermopneumatically actuatedpolydimethylsiloxane (PDMS) valve is used to control flow into and outof the chamber(s) (see FIG. 9H). In this embodiment, the valve isconstructed using multi-layer PDMS microfabrication (see FIG. 9I). Thevalve is opened and closed using a liquid that upon the application ofheat causes the fluid to expand, deforming the membrane, thus blockingaccess to the chamber. Preferably, a liquid that exhibits a low boilingpoint and a range of vapor pressures at different temperatures, such asVertrel XF™ is used. Vertrel XF™ (2,3,dihydrodecafluoropentane-C₅H₂F₁₀;available from DuPont), is a hydrocarbon fluid that has a low boilingpoint (55° C.) and exerts vapor pressures ranging from 4.4 psi (at 25°C.) to 17.8 psi (at 60° C.). An external peltier device may be placedbelow the vertrel chamber to heat or cool the vertrel.

In other embodiments, piezoelectric (PZT) mixers are used as valves.These can be built out of silicon (obtained from Frauhoffer), plastic(obtained from IMM) or PCB.

Other materials can be used in combination with check valves includematerials that can be used to block an inlet or an outlet port. Suchmaterials include wax or other polymeric materials, such aspoly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblockcopolymers (PEO-PPO-PEO) known commercially as Pluronics (BASF; PluronicF-127, Sigma) or Synperonic (ICI), that melt for use as membranes orplugs. These materials share the common feature that they can go from asolid to a liquid at a given temperature. These types of systems areused in conjunction with heaters, described below. For example, heat isapplied to melt the material, thus “opening” the valve.

In a preferred embodiment, valves may be constructed out of any materialthat is a solid at room temperature or at an assay temperature (e.g.,temperatures used in PCR reactions). Suitable materials includepolymers, waxes, paraffin, gels, agarose, etc.

In a preferred embodiment, paraffin (i.e., wax) valves are used tocontrol flow into and out of the chamber. Paraffin valves may be singlecycle or multicycle valves. Examples of single cycle valves include“close-open”, “open-close-open”, and “T” valves. An example of amulticycle valve is a “toggle” valve.

In a preferred embodiment, “close-open” paraffin valves are used (seeFIG. 9J). In this embodiment, the valve uses a bulk of paraffin that islocalized in a heating zone to close the channel. To prevent the waxfrom blocking the downstream channel after the valve is turned on, thedownstream channel may designed with a wider section so that the wax cansolidify without blocking the channel.

In a preferred embodiment, “open-close-open” paraffin valves are used(see FIG. 9K). In this embodiment, the valve is open and is closed bymelting the paraffin. The valve is reopened using an air pump to drivethe molten wax into the downstream channel.

In a preferred embodiment, “T” valves are used (see FIG. 9L). In thisembodiment, the microfluidic device contains a “T” channel in which theentrance to each branch is gated with a paraffin valve. Flow is directedin the center channel down one branch or the other depending on whichvalve is open and which valve is closed. As shown in the FIGS. 9L(1) and(2), the branch on the left side is gated with an “open-close” valve,while the branch on the right side is gated with a “close-open” valve.In the absence of heat, fluid moves down the center and left channels.When heat is applied to both valves, the valve on the left side isclosed and the valve on the right side is opened, thus directly fluidflow down the center and right channels.

In a preferred embodiment, “toggle” valves are used (see FIG. 9M).Toggle valves consist of two or more “open-close-open” valves.

In a preferred embodiment, valves are made using Pluronics. Pluronics,are surfactant materials that are liquids at low temperatures and solidsat high temperature (i.e., materials that aggregate to form micellesabove a critical concentration). For example, solutions of Pluoronicswithin a concentration range of 18-30% are low viscosity liquids at lowtemperature (0-5° C.), but form self-supporting cubic liquid crystallinegels at room temperature. Pluronic valves may be incorporated into themicrofluidic devices of the current invention by injecting the Pluronicliquid gel into a microchannel (see FIG. 9N).

In a preferred embodiment, the burst valve is a film of metal orpolymer. In a preferred embodiment, a free standing gold film is used,that is constructed using standard techniques as outlined herein, byetching away a support surface. The gold membrane dissolves uponapplication of a voltage and Cl⁻ ions. See for example www.mchips.com;Santini, J. T., et al., 1999, Nature, 397:335-338; both of which areincorporated by reference in their entirety.

In a preferred embodiment, a combination of check valves and wax plugsare used. In other embodiments, a combination of check valves and goldmembranes are used.

Other means of making a valve include mechanical means. These canfrequently be bi-directional valves. For example, a shape memory wirecan be attached to a plunger blocking a channel. By applying a currentto the wire, the wire contracts and moves the plunger out of the way,thereby opening the channel. Conversely, the plunger can be drawn intothe channel to block the channel. See FIG. 9C.

Other mechanical valves include rotary valves. Rotary valves can beconfigured in a variety of ways, as depicted in FIGS. 9D and 9E. In oneembodiment, an external force must be applied for rotation (i.e., ascrew driver or stepper motor). Alternatively, a shape memory wire canbe used, such that the application of heat or current will shrink thewire to rotate the valve. See FIG. 9E.

In addition, commercially available valves may be used in to control theflow of liquids from into and out of the various chambers of the presentinvention. Examples of commercially available valves include, MEMS(micro-electro-mechanical systems) micro valves(http://www.redwoodmicro.com), TiNi liquid microvalve (TiNi AlloyCompany, San Leandro, Calif.), TiNi pneumatic microvalves (TiNi AlloyCompany, San Leandro, Calif.), silicon micro valves (Bosch, D., et al.,Sensors and Activators A, 37-38 (1993) 684-692). Commercial/conventionalvalves also are available from Measurement Specialities, Inc., ICSensors Division, Milpitas, Calif. (http://www.msiusa.com/icsensors);Plast-O-Matic Valves, Inc. (http://www.plastomatic.com/), Barworth Inc.(http://www.barworthinc.com), Mobile Electronics Solution(http://www.mobileelectronics.net/); Specrum Chromatograph(http://www.lplc.com); all of which are hereby incorporated by referencein their entirety.

Other sources for obtaining valves and pumps include researchfoundations such as Chronos (http://www.memsrus.com); Institute forMicrotechnology—Mainz (http://www.imm-mainz.de/); Microsystemsintegration group—Swiss Fed. Inst. of technology(http://dmtwww.epfl.ch); University of Washington(http://lettuce.me.washington.edu); The Berkeley Sensor and ActuatorCenter (BSAC) (http://www.otl.berkeley.edu/mems.html); University ofMichigan, Microsystems R&D Laboratory(http://www.eecs.umich.edu/MEMS/facilities.html); Caltech, MEMS Research(http://touch.caltech.edu/home/research/files/html/researchframe.html);all of which are hereby incorporated by reference in their entirety.

In a preferred embodiment, either an “on chip” or “off chip” pump isused to move fluids from one area or chamber of the cartridge toanother. A general design for a pump includes a chamber through whichair and/or liquid can flow; an inlet and outlet port, and valves. Fluidis moved through the pump via the application of some force, such asheat, pressure, to the chamber. In addition, pumps may be designed forsingle use or be reusable. Generally, reusable pumps have valves, i.e.,check valves, that bias the flow of fluid in one direction. Single usepumps lack valves. Thus, almost any type of pump can be built as long asa mechanism for changing the volume in the chamber and restricting backflow is included. For example, upon contraction of the chamber via a PZTor other pressure force, the fluid or gas is displaced out of thechamber through the check valve. Upon removal of the contractive force,the chamber expands and draws liquid in through the pump inlet.Alternatively, a heater can be placed inside the chamber and thetemperature of the gas or liquid can be raised, causing it to expand.Upon expansion, the liquid is forced out of the chamber through theoutlet. Upon cooling the liquid, the fluid is draw in through the inlet.See FIG. 9G.

There are two primary means by which fluid can be moved in the biochipcartridge. These are: (1) through the use of a pump that pushes thefluid in or out; or, (2) by suction that pulls fluid in or out of thechamber.

Generally, a device such as a moving piston is used to create suction,however cooling of gases, vacuum chambers and gas consuming reactionscan be used. When suction is used to move liquid in or out of thechamber, a vacuum may be created elsewhere in the system.

Basically, two major groups of pumps, classified based on different pumpmechanisms (i.e., actuation), can be use in the present invention:membrane actuated (i.e., mechanical) and non-membrane actuated pumps.Membrane actuated pumps can be further divided into three types:piezoelectric, electrostatic, and thermopneumatic. Non-membrane pumpingprinciples include electrohydrodynamic, electroosmotic, traveling wave,diffuser, bubble, surface wetting, rotary, etc.

In a preferred embodiment, an “air pump” is used to move the liquid outof the PCR chamber. In this embodiment, a chamber of air is incorporatedin the chip with an “on chip” heater. When the heater is turned on, theair in the chamber expands according to PV=nRT. In some embodiments, theair pump is incorporated into the cartridge.

Preferably, heaters (as are also described below) are incorporated intothe middle of the chip. In some embodiments, more than one heater isincorporated in a chip to create “heater zones”. Air chambers or pocketsare located over the heater zones. The air chambers are connected to thereaction chamber via a channel that runs up to the top of the reactionchamber with a valve or a plug blocking it off. When the air is heated,it expands. The resulting build up in pressure forces the valve or plugto move out of the way, thereby forcing the liquid out of the chambervia an outlet port.

Other ways of moving liquid out of the reaction chamber or reactionchamber include using a low boiling liquid in place of air. In thisembodiment, the low boiling liquid expands when heated and displaces theliquid contained in the reaction chamber. Alternatively, a chemicalreaction may be used to move liquid out of the reaction chamber. Forexample, the chemical reaction used to expand car air bags may be usedto move liquid out of the reaction chamber, or other reactions in whichgases are generated.

Other types of pumps that can be used include syringe driven pumps.These pumps can be actuated either by expanding air behind the syringeor by mechanical means. For example, TiNi alloys, nitinol wire, or“shape memory metals” can be used to mechanically actuate a syringedriven pump. By “TiNi alloys”, “nitinol wire” or “shape memory metals”herein is meant materials that when heated above a certain transitiontemperature contract (i.e., usually up to 3 to 5% over the originallength of the metal), thereby changing shape. Other materials thatchange shape upon heating include shape memory plastics.

Pumps also may be created using spring loaded pistons. In thisembodiment, a spring that can be released is compressed or restrainedwithin the body of the cartridge. For example, wax may be used to hold aspring in its compressed state. Upon heating, the wax is melted, and thespring is released, thereby generating sufficient force to move a pistonand displace liquid. Other versions include incorporating materials thatchange from solids to liquids at a given transition temperature, ormoving a mechanical blockade from the spring's pathway.

Pumps that utilize PZT driven actuations are also known and may beincorporated into this invention. By “PZT” herein is meant a materialcomprised of lead, zirconium and titanium which upon application of avoltage undergoes a rearrangement of the crystal lattice and generates aforce and a displacement. This so called piezoelectric effect can beused to constrict and expand a pump chamber and result in a net movementof liquid. Other materials like shape memory alloys that under a changein shape upon application of a current such that the temperature of themetal is raised above a certain transition temperature can also be used.

In addition, commercially available micro pumps may be used in to moveliquid from one location to another in the cartridge. Examples ofcommercially available pumps include, moulded plastic micro pumpsavailable from IMM (see liganews@imm.uni-mainz.de), thin film shapealloy microactuators (TiNi Alloy Company, San Leandro, Calif.), siliconmicro pumps (see M. Richter & J. Kruckow,aktorik/paper/2000_jahresbericht/Paper2, 16.11.00).

In addition, based on the geometry of the chamber, air can be used topush liquid out of the reaction chamber or mix liquids within thereaction chamber. Whether the air pumps the fluid or bubbles through togenerate a mixing effect is determine by the relative size of thebubble, the geometry of the chamber/channel and the surface tension ofthe liquid. Larger air-liquid interfaces tend to favor mixing overpumping. Mixing of liquid within the biochip cartridge can occur bypumping the liquid back and forth in the biochip cartridge.

In a preferred embodiment, flow-induced mixing is used to induceconvectional flow. Preferably, this is used in a vertical system, suchthat fluid gravity may be used to induce convectional flow. Theconvectional flow results in bulk fluid mixing between two liquidsolutions. In addition, meniscus recirculation mixing can be used toinduce circulation flow (Anderson, et al., (1998) Solid-State Sensor andActuator Workship, Hilton Head Island, June 9-11, pp 7-10; incorporatedherein by reference in its entirety).

In a preferred embodiment, mixing is used to enhance hybridizationrates. In a preferred embodiment, mixing is accomplished by inducingacoustic streaming using a piezoelectric transducer glued onto the backof the cartridge and excited with 5 Khz a.c. waveform at 10 V_(p-p).

In a preferred embodiment, mixing is accomplished by creating a thermalgradient across a chip. For example, a thermal gradient may be createdby heating the bottom of the chip to 65° C. and cooling the top of thecartridge cover to 10° C. This can be accomplished by placing the chipbetween two peltier heaters, or by using an imbedded heater and a singlepeltier or other thermoelectric cooling devices.

In a preferred embodiment, mixing is accomplished by recirculatingliquid in a given chamber using an on chip or “off chip” pump attachedto a chip.

In alternative embodiments, mixing is accomplished by recirculatingliquid using a micro disk-pump, such as a plastic disk embedded with amagnetic steel bar. Rotation of the disk pump may be achieved using anexternal magnetic filed provided by a standard stirrer or custom builtwith multiple fields. See also U.S. Ser. No. 60/308,169, filed Jul. 26,2001 and a provisional application by Gallagher, et al., entitled“System and methods for mixing within a microfluidic chamber”, filedJul. 11, 2002; both of which are incorporated by reference in theirentirety.

In other embodiments, biochannel based mixing can be used to enhancehybridization rates. In this embodiment, a bubble is intentionallyintroduced into one corner of the chip. By alternately expanding andcontracting the bubble volume via the application of heat from either anin chip or off chip heat source, mixing occurs as a result of thepressure flow created by changing the volume of the bubble within thechip. Alternatively, resonance induced mixing of bubbles can be doneusing PZT devices as well.

In some embodiments, mixing may be accomplished using non-contact mixingtechnologies like that describe by Covaris, Inc.

In a preferred embodiment, heaters are incorporated onto or into thechip, to allow “on chip” heating (in addition, as described below, “offchip” thermocontrollers within the device may also be used). In thisembodiment, the reaction chamber is designed to maximize thermalconductivity between the chamber and the heater or thermocontroller.Generally, designs that minimize thermal mass (i.e., making the surfaceof the chamber in contact with the heat source as thin as possible),impose certain geometric constraints to ensure the complete removal ofliquid from the chamber, incorporate materials that are good thermalconductors (i.e., metals), and thermally isolate the chamber from therest of the chip are preferred. Often one makes a trade off betweenminimizing surface to volume ratios to reduce surface area for thenon-specific binding of biological components and maximizingsurface-to-volume ratis in order to obtain rapid heat transfer rates forheating and cooling.

In a preferred embodiment, air pockets or vents are used to thermallyisolate the amplification chamber from the rest of the chip. That is,the there is a break in the continuity of the cartridge around theamplification chamber.

In a preferred embodiment, thermally conductive materials areincorporated into or below the reaction chamber, forming hybridchambers. For example, by using “layers” of different materials,effective heaters are constructed. Thus for example, a preferredembodiment utilizes one or more resistive heaters in the form ofresistive metallic inks can be applied to a first layer of PC board.These heaters are powered by interconnects. In a preferred embodiment, athin sheet of a thermally conductive material, preferably a metal suchas copper, is applied, to allow even heat distribution. In a preferredembodiment, the copper layer is then coated with a thin layer ofbiocompatible material, such as plastic. See FIG. 10A.

The total thickness of the hybrid chamber may vary from a few microns tomillimeter dimension. A preferred thickness is approximately 200microns.

In a preferred embodiment, multiple thermal heaters are incorporatedinto the device to allow for the creation of multiple thermal zones. Thetemperature in the respective zones is maintained via either active orpassive control. Frequently, the thermal connectivity of the cartridgematerials are taken into account during the design. In one embodiment, achip may contain a thermal heater in the detection chamber of thecartridge in order to maintain the temperature of the detection chamberas well as constructing unique temperature zones in another part ofdevice. In one embodiments, these temperature zones may be maintained toallow an enzymatic reaction to run efficiently. In another embodiment,multiple temperature zones may be maintained to simulate thetemperatures normally used during PCR heat cycling. In order to effectthe necessary temperature, the liquid can be maintained stationary andthe temperature of the amplification chamber cycled (i.e. 95-55-72),alternatively, the liquid can be pumped over different temperature zonesin order to obtain heat cycling (FIG. 10 B). This embodiment can berealized in different material substrates such as glass, plastic,ceramic and PCB.

Similarly, there may be portions of the substrate that require heating,and those that do not. Thus more than one heater may be incorporatedinto the substrate. Similarly, these thermal zones may or may not bethermally isolated from other parts of the substrate. For example, PCboard is significantly thermally insulative, and thus just puttingdistance between the heaters and thermal zones and the areas of thesubstrate that do not require heating may be sufficient. In otherembodiments, thermally insulative materials may be incorporated. Forexample, when the substrate is a ceramic material, thermal isolation maybe accomplished by cutting out sections of the ceramic substrate suchthat solid regions of ceramic are separated from one another by a “cutout” as shown in FIG. 10C.

Other embodiments include the incorporation of temperature sensors intothe substrate such that the temperature throughout the board can bemonitored. In a preferred embodiment, temperature sensors are createdusing resistive devices, including silicon diodes. Other embodimentsinclude the use of capillary thermostats and limiters(http://www.thermodisc.com/BulbAndCapillary.html).

As will be appreciated by those in the art, there are a variety ofreaction chamber geometries which can be used in this way. Generallyhaving the intersection of the inlet port and the reaction chamber be atthe “bottom” of the cartridge, with a small aperture, with the reactionchamber widening, is preferred. In addition, the “top” of the reactionchamber may narrow, as well. Several embodiments are depicted in FIG. 2.Thus, preferred embodiments for the size and shape of the reactionchamber allow for smooth loading of the reaction chamber. Preferredembodiments utilize reaction chamber geometries that avoid the use ofsharp corners or other components that serve as points for bubbleformation.

In addition, in some embodiments, the reaction chamber can be configuredto allow mixing of the sample. For example, when a sample and a reagentare introduced simultaneously or separately into the chamber, the inletport and/or the reaction chamber can comprise weirs, channels or othercomponents to maximize the mixing of the sample and reagent. Inaddition, as is outlined below, the reaction may utilize magnetic beadsfor mixing and/or separation.

In a preferred embodiment, the cartridge comprises a sealing and/orventing mechanism to prevent the cartridge from exploding due to a buildup in pressure during a reaction, or to prevent leakage of the sample orreagents onto other parts of the substrate, particularly (in the case ofelectronic detection) onto electronic interconnects. As will beappreciated by those in the art, this may take on a variety of differentforms. In one embodiment, there is a gasket between the biochipsubstrate comprising the array and the cartridge, comprising sheets,tubes or strips. Alternatively, there may be a rubber or silicone stripor tube used; for example, the housing may comprise an indentation orchannel into which the gasket fits, and then the housing, gasket andchip are clamped together. Furthermore, adhesives can be used to attachthe gasket to the cartridge, for example, a double sided adhesive can beused; for example, silicone, acrylic and combination adhesives can beused to attach the gasket to the biochip, which is then clamped into thecartridge as described herein.

In some embodiments, the reaction chamber and biochip substrate areconfigured such that a separate sealing mechanism is not required. Forexample, the biochip substrate can serve as one “half” of the reactionchamber, with the array on the inside, and the reaction chamber housingcan serve as the other “half”. Depending on the materials used, theremay be an optional adhesive to attach the two. Alternatively, when thereare arrays on both sides of the substrate, the housing may encompass thesubstrate.

Optional adhesives that may be used to seal the cartridge include butare not limited to, pressure sensitive adhesives, thermal adhesives, amulti-layer adhesive formed by overlaying double-sided tape with Teflon™core with a Parafilm™ layer and then covering the Parafilm™ layer, etc.Other means of sealing the cartridges include sonic welding, solventassisted thermal bonding, laser bonding, and epoxys.

In a preferred embodiment, the reaction chamber is made entirely ofplastic. In another embodiment, a PCB underlies all or a significantportion of the cartridge. The cartridge may be attached directly to thePCB. Alternatively, the device can be built wholly in the PCB, ceramic,or glass material with all of the necessary or a large majority of thenecessary functions integrated into the device during the manufacturingprocess.

Thus, in these embodiments, the volume of the reaction chamber can beset either by forming a well in the cartridge, such that the addition ofthe biochip substrate forms a reaction chamber around the array, or byusing a flat cartridge and using a gasket or adhesive of a defineddepth, or by combinations of the three.

In a preferred embodiment, the cartridge comprises a cap or lid. The capmay be functional, as outlined below when it comprises microfluidiccomponents. In addition, the cap may be designed for safety purposes, toprevent the leakage of biological materials or cross-contamination.Additionally, the cap can be designed to be removable. As will beappreciated by those in the art, the cap can take on a wide variety ofconfigurations. For example, in one embodiment, the cap merely seals theinlet port to prevent evaporation of the sample during the assay. In apreferred embodiment, the cap may comprise a number of additionalelements for use in sample handling and reagent storage, to allow for avariety of different sample reactions. For example, a variety ofmicrofluidic components can be built into the cap to effect a number ofmanipulations on a sample to ultimately result in target analytedetection or quantitation. See generally PCT US00/10903, and referencesoutlined therein, all of which are expressly incorporated by reference.These manipulations can include cell handling (cell concentration, celllysis, cell removal, cell separation, etc.), separation of the desiredtarget analyte from other sample components, chemical or enzymaticreactions on the target analyte, detection of the target analyte, etc.The devices of the invention can include one or more wells for samplemanipulation, waste or reagents; microchannels (sometimes referred to asflow channels) to and between these wells, including microchannelscontaining electrophoretic separation matrices; valves to control fluidmovement; on-chip pumps such as electroosmotic, electrohydrodynamic, orelectrokinetic pumps. In addition, as outlined herein, portions of theinternal surfaces of the device may be coated with a variety of coatingsas needed, to reduce non-specific binding, to allow the attachment ofbinding ligands, for biocompatibility, for flow resistance, etc. Thesemicrofluidic caps can be made in a variety of ways, as will beappreciated by those in the art. See for example references described inPCT US00/10903, and references outlined therein, all of which areexpressly incorporated by reference.

When the cap of the cartridge is used as part of the assay, it may beconfigured to include one or more of a variety of components, hereinreferred to as “modules”, that will be present on any given devicedepending on its use, and are connected as required by microchannels.These modules include, but are not limited to: sample inlet ports;sample introduction or collection modules; cell handling modules (forexample, for cell lysis, cell removal, cell concentration, cellseparation or capture, cell growth, etc.); separation modules, forexample, for electrophoresis, dielectrophoresis, gel filtration, ionexchange/affinity chromatography (capture and release) etc.; reactionmodules for chemical or biological alteration of the sample, includingamplification of the target analyte (for example, when the targetanalyte is nucleic acid, amplification techniques are useful, including,but not limited to polymerase chain reaction (PCR), oligonucleotideligation assay (OLA); strand displacement amplification (SDA), andnucleic acid sequence based amplification (NASBA) and other techniques'outlined in WO 99/37819, PCT US00/19889, and US00/20476, all of whichare hereby incorporated by reference in their entirety; chemical,physical or enzymatic cleavage or alteration of the target analyte, orchemical modification of the target; fluid pumps (including, but notlimited to, electroosmotic, electrohydrodynamic, or electrokineticpumps; fluid valves; thermal modules for heating and cooling; storagemodules for assay reagents; mixing chambers; and detection modules.

In addition, while these microfluidic components are described herein asbeing associated with the cap of the cartridge, as will be appreciatedby those in the art, these modules and channels (as well as othercomponents outlined herein) may be located anywhere in the cartridge ordevice. In addition, some components may be in the device; for example,“off chip” pumps may be located within one or more stations of thedevice.

The cartridge comprises at least one biochip, with some embodimentsutilizing one or more biochips per cartridge. By “biochip” orequivalents herein is meant a substrate comprising an array of distinctbiomolecules, particularly nucleic-acids and proteins. There are a widevariety of suitable nucleic acid biochips, including those made usingphotolithographic techniques (such as the Affymetrix GeneChip™),spotting techniques (e.g. Synteni and Incyte), prining techniques(Agilent and Rosetta), three dimensional “gel pad” arrays, and thoseincluding electronic components (e.g. Nanogen). A preferred embodimentis described below and in U.S. Pat. Nos. 5,591,578; 5,824,473;5,705,348; 5,780,234 and 5,770,369; U.S. Ser. Nos. 08/873,59808/911,589; WO 98/20162; WO98/12430; WO98/57158; WO 00/16089)WO99/57317; WO99/67425; WO00/24941; PCT US00/10903; WO00/38836;WO99/37819; WO99/57319 and PCTUS00/20476; and related materials, all ofwhich are expressly incorporated by reference in their entirety.

It should be noted that one distinct advantage of the use of theelectronic detection methods outlined herein is that real timemonitoring of reactions and hybridization can occur. That is, whilesystems based on fluorescence require the removal of excess (e.g.unbound) signaling probes (or target sequences when the target sequenceitself has been fluorescently labeled during an amplification reaction,for example), the electronic methods outlined herein do not. That is,unless the probes comprising the ETMs are bound to the surface, littleor no signal is seen even if unbound probes have not been removed. Thisallows the monitoring of real-time reactions, as well as multiplemeasurements on the same array. Accordingly, while the discussion belowis directed mainly to the use of biochips comprising an array ofelectrodes, other array technologies are included in the presentinvention.

In a preferred embodiment, the biochips comprise substrates with aplurality of array locations. By “substrate” or “solid support” or othergrammatical equivalents herein is meant any material that can bemodified to contain discrete individual sites appropriate of theattachment or association of capture ligands. Suitable substratesinclude metal surfaces such as gold, electrodes as defined below, glassand modified or functionalized glass, fiberglass, teflon, ceramics,mica, plastic (including acrylics, polystyrene and copolymers of styreneand other materials, polypropylene, polyethylene, polybutylene,polyimide, polycarbonate, polyurethanes, Teflon™, and derivativesthereof, etc.), GETEK (a blend of polypropylene oxide and fiberglass),etc, polysaccharides, nylon or nitrocellulose, resins, silica orsilica-based materials including silicon and modified silicon, carbon,metals, inorganic glasses and a variety of other polymers, with printedcircuit board (PCB) materials being particularly preferred.

The present system finds particular utility in array formats, i.e.wherein there is a matrix of addressable detection electrodes (hereingenerally referred to “pads”, “addresses” or “micro-locations”). By“array” herein is meant a plurality of capture ligands in an arrayformat; the size of the array will depend on the composition and end useof the array. Arrays containing from about 2 different capture ligandsto many thousands can be made. Generally, the array will comprise fromtwo to as many as 100,000 or more, depending on the size of theelectrodes, as well as the end use of the array. Preferred ranges arefrom about 2 to about 10,000, with from about 5 to about 1000 beingpreferred, and from about 10 to about 100 being particularly preferred.In some embodiments, the compositions of the invention may not be inarray format; that is, for some embodiments, compositions comprising asingle capture ligand may be made as well. In addition, in some arrays,multiple substrates may be used, either of different or identicalcompositions. Thus for example, large arrays may comprise a plurality ofsmaller substrates.

In a preferred embodiment, the biochip comprises a substrate with atleast one surface comprising an array, and in a preferred embodiment, anarray of electrodes. By “electrode” herein is meant a composition,which, when connected to an electronic device, is able to sense acurrent or charge and convert it to a signal. Alternatively an electrodecan be defined as a composition which can apply a potential to and/orpass electrons to or from species in the solution. Thus, an electrode isan ETM as described herein. Preferred electrodes are known in the artand include, but are not limited to, certain metals and their oxides,including gold; platinum; palladium; silicon; aluminum; metal oxideelectrodes including platinum oxide, titanium oxide, tin oxide, indiumtin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenumoxide (Mo2O6), tungsten oxide (WO3) and ruthenium oxides; and carbon(including glassy carbon electrodes, graphite and carbon paste).Preferred electrodes include gold, silicon, carbon and metal oxideelectrodes, with gold being particularly preferred.

The electrodes described herein are depicted as a flat surface, which isonly one of the possible conformations of the electrode and is forschematic purposes only. The conformation of the electrode will varywith the detection method used and the configuration of the cartridge.For example, flat planar electrodes may be preferred for opticaldetection methods, or when arrays of nucleic acids are made, thusrequiring addressable locations for both synthesis and detection.Alternatively, for single or low density analysis, the electrode may bein the form of a tube; this allows a maximum of surface area containingthe nucleic acids to be exposed to a small volume of sample.

In a preferred embodiment, the detection electrodes are formed on asubstrate. In addition, the discussion herein is generally directed tothe formation of gold electrodes, but as will be appreciated by those inthe art, other electrodes can be used as well. The substrate cancomprise a wide variety of materials, as outlined above.

In general, preferred materials include printed circuit board materials.Circuit board materials are those that comprise an insulating substratethat is coated with a conducting layer and processed using lithographytechniques, particularly photolithography techniques, to form thepatterns of electrodes and interconnects (sometimes referred to in theart as interconnections or leads). The insulating substrate isgenerally, but not always, a polymer. As is known in the art, one or aplurality of layers may be used, to make either “two dimensional” (e.g.all electrodes and interconnections in a plane) or “three dimensional”(wherein the electrodes are on one surface and the interconnects may gothrough the board to the other side or wherein electrodes are on aplurality of surfaces) boards. Three dimensional systems frequently relyon the use of drilling or etching, followed by electroplating with ametal such as copper, such that the “through board” interconnections aremade. Circuit board materials are often provided with a foil alreadyattached to the substrate, such as a copper foil, with additional copperadded as needed (for example for interconnections), for example byelectroplating. The copper surface may then need to be roughened, forexample through etching, to allow attachment of the adhesion layer.

Accordingly, in a preferred embodiment, the present invention providesbiochips (sometimes referred to herein “chips”) that comprise substratescomprising a plurality of electrodes, preferably gold electrodes. Thenumber of electrodes is as outlined for arrays. Each electrodepreferably comprises a self-assembled monolayer as outlined herein. In apreferred embodiment, one of the monolayer-forming species comprises acapture ligand as outlined herein. In addition, each electrode has aninterconnection, that is attached to the electrode at one end and isultimately attached to a device that can control the electrode. That is,each electrode is independently addressable.

In a preferred embodiment, the connections from the electrodes are madeby passing through the substrate to produce a so called land grid arraythat can interface to a pogo pin or like connector to make connectionsfrom the chip to the instrument. In this embodiment, pogo pin connectorsare used in place of edge card connectors. An example of a chipcontaining electrodes arranged in a land grid array is shown in FIG. 11.In this embodiment, rather than contain longer interconnects, theelectrode array is one surface of the substrate, such as a PCR board orceramic substrate, and there are “through board” or “through substrate”interconnects ending in pads. See FIG. 1J. When the cartridge is placedin the device, these pads contact “pogo pin” type connectors, thussaving space on the chip and allowing for higher density arrays, ifdesired. See FIG. 1K. In some embodiments, switching circuitry(multiplexers) can be built into the pogo pin connector.

Detection electrodes on circuit board material (or other substrates) aregenerally prepared in a wide variety of ways. In general, high puritygold is used, and it may be deposited on a surface via vacuum depositionprocesses (sputtering and evaporation) or solution deposition(electroplating or electroless processes). When electroplating is done,the substrate must initially comprise a conductive material; fiberglasscircuit boards are frequently provided with copper foil. Frequently,depending on the substrate, an adhesion layer between the substrate andthe gold in order to insure good mechanical stability is used. Thus,preferred embodiments utilize a deposition layer of an adhesion metalsuch as chromium, titanium, titanium/tungsten, tantalum, nickel orpalladium, which can be deposited as above for the gold. Whenelectroplated metal (either the adhesion metal or the electrode metal)is used, grain refining additives, frequently referred to in the tradeas brighteners, can optionally be added to alter surface depositionproperties. Preferred brighteners are mixtures of organic and inorganicspecies, with cobalt and nickel being preferred.

In general, the adhesion layer is from about 100Δ thick to about 25microns (1000 microinches). The If the adhesion metal iselectrochemically active, the electrode metal must be coated at athickness that prevents “bleed-through”; if the adhesion metal is notelectrochemically active, the electrode metal may be thinner. Generally,the electrode metal (preferably gold) is deposited at thicknessesranging from about 500Δ to about 5 microns (200 microinches), with fromabout 30 microinches to about 50 microinches being preferred. Ingeneral, the gold is deposited to make electrodes ranging in size fromabout 5 microns to about 5 mm in diameter, with about 100 to 250 micronsbeing preferred. The detection electrodes thus formed are thenpreferably cleaned and SAMs added, as is discussed below.

Thus, the present invention provides methods of making a substratecomprising a plurality of gold electrodes. The methods first comprisecoating an adhesion metal, such as nickel or palladium (optionally withbrightener), onto the substrate. Electroplating is preferred. Theelectrode metal, preferably gold, is then coated (again, withelectroplating preferred) onto the adhesion metal. Then the patterns ofthe device, comprising the electrodes and their associatedinterconnections are made using lithographic techniques, particularlyphotolithographic techniques as are known in the art, and wet chemicaletching. Frequently, a non-conductive chemically resistive insulatingmaterial such as solder mask or plastic is laid down using thesephotolithographic techniques, leaving only the electrodes and aconnection point to the leads exposed; the leads themselves aregenerally coated.

In one embodiment of the inventive structure, the solder mask isdesirably made of a solvent soluble material rather than a water solublematerial. Water soluble solder masks have become standard in theindustry because of the environmental advantages of water solublematerials generally. Unfortunately, for a detector chip that is to beexposed to aqueous solutions, water soluble materials such as forexample acetonitrile can dissolve when exposed to aqueous solution.

The methods continue with the addition of SAMs, described below. In apreferred embodiment, drop deposition techniques are used to add therequired chemistry, i.e. the monolayer forming species, one of which ispreferably a capture ligand comprising species. Drop depositiontechniques are well known for making “spot” arrays. This is done to adda different composition to each electrode, i.e. to make an arraycomprising different capture ligands. Alternatively, the SAM species maybe identical for each electrode, and this may be accomplished using adrop deposition technique or the immersion of the entire substrate or asurface of the substrate into the solution.

In a preferred embodiment, plasma treatments are used to generate asurface free of major contaminants prior to the deposition of SAMscomprising capture probes. This method is particularly useful for theactivation of gold surfaces for the formation of SAMs comprising captureprobes with packing densities close to the theoretical limit (see FIG.68). Plasma methods can also be used for the deposition of differentcapture probes on neighboring pads.

Plasma treatment in a barrel type machine.(minimal ion bombardment) withoxygen plasma is commonly used in semiconductor processing to removetrace residues of organic contaminants, including photoresistantcontaminants. Although treatment with oxygen plasma can be used in themethods of the present invention for generating a clean surface, thistreatment makes the insulator layer separating the gold padshydrophilic. Thus, it is difficult to spot arrays without contamination.

In a preferred embodiment, a hydrogen plasma treatment is used. Thisprocedure recovers hydrophobicity on organic surfaces because itconverts hydrophilic C—O bonds to C—H bonds. In addition, this treatmentdoes not add contaminants to the gold surface. Hydrogen plasma treatmentcan be used alone, or in combination with an oxygen plasma treatment.FIG. 69 illustrates the effectiveness of an oxygen plasma treatmentfollowed by a hydrogen plasma treatment for removing major contaminantsfrom chips.

Combining an oxygen plasma treatment with a hydrogen plasma treatmentcan be used on micro-patterned photoresistant materials to generatehydrophobic surfaces next to hydrophilic surfaces. The hydrophobic andhydrophilic surfaces can either be adjacent to each other or separatedby a few microns.

In a preferred embodiment, CF₄ plasma can be used after an oxygen plasmatreatment to make Teflon™-like compounds, i.e. C—F bonds, on theinsulator, resulting in a hydrophobic insulator surface.

In other embodiments, a chemical treatment can be combined with a plasmatreatment. For example, chemical treatment with silanes can be used tomake the oxide insulator surface hydrophobic or hydrophilic, whileleaving non-oxide surfaces, such as gold, uncontaminated. Whether theinsulator surface is hydrophobic or hydrophilic depends on the type ofsilane that replaces the O—H bonds. Additionally, this procedure can beused in conjunctions with plasma cleaning to enable selective wetting ofchemical and biological fluids on the chip surface or to prevent orallow sticking of a given species of solution to the surface of thechip.

In a preferred embodiment, plasma treatments are used to generate apattern of hydrophobic surfaces adjoining hydrophilic surfaces on achip. Generation of hydrophobic insulating surfaces between neighboringelectrodes prevents electrodes with different DNA capture probes fromcontaminating each other. As described above, oxygen plasma treatmentscan be used to generate a hydrophilic insulator surface on a chip.Hydrogen plasma treatments may be used to generate a hydrophobicinsulator surface. Thus, combinations of oxygen plasma treatmentsfollowed by a hydrogen plasma treatment or a CF₄ plasma treatment can beused to form a pattern of hydrophobic surfaces adjoining hydrophilicsurfaces.

In a preferred embodiment, a hydrogen plasma treatment is used incombination with an oxygen plasma treatment to generate a pattern ofhydrophobic surfaces adjoining hydrophilic surfaces.

In some embodiments, a hydrogen plasma treatment is used to generate ahydrophobic surface next another hydrophobic surface.

In some embodiments, a CF₄ plasma treatment is used in combination withan oxygen treatment to make teflon-like compounds (i.e., C—F bonds) onthe hydrophilic insulator surface, thereby generating a pattern ofhydrophilic surfaces adjoining hydrophobic surfaces.

In other embodiments, chemical treatments with silanes can be used tomake the oxide insulator surfaces hydrophobic or hydrophilic dependingon the type of silane that replaces the O—H bonds in the aqueoussolution. This same procedure can be used in conjunction with plasmacleaning to allow selective wetting of chemical and biological fluidswith the chip surface to either prevent or allow sticking of a givenspecies to the chip surface.

When the biochips comprise electrodes, there are a variety of additionalcomponents in addition to the chemistry outlined below, which may bepresent on the chip, including, but not limited to, interconnects,multiplexers, relay devices, filters, RF antennae, heating elements,electromagnetic components, etc.

In some embodiments, antenna can be incorporated into the chip.Preferably, at least one antennae is used per electrode array. Antennasmay be either inductive or capacitive (see FIGS. 74(A) and (B). Theantenna may be designed to transmit independently or respond to a signalfrom the multiplexing device.

Each electrode comprises an independent lead (interconnect) to transmitinput and electronic response signals for each electrode of the array.In contrast to previous systems which require the ability toindependently alter only input signals to each electrode but notelectronic response signals, it is important in the present inventionthat both input and electronic response signals be independentlymonitorable for each electrode.

For a relatively small number of electrode pads and/or depending on thedesired size of the array, providing direct connections using parallelcircuits may be appropriate.

In a preferred embodiment, each electrode is individually connected to acorresponding input of a multiplexer via a corresponding interconnector.One problem presented in conventional systems and methods is thedifficulty in providing electrical connections (inputs and/or outputs)to a large number of electrodes, particularly if the electrodes form adense or close packed array. Several solutions to this problem have beenidentified, and include the use of circuitry that allows signalprocessing either simultaneously as sets of parallel circuits andconnections, line-sample array addressing, serially in a time-domainmultiplexed manner, or in parallel or serially using frequency domainand/or time-domain based separation techniques, among other availabletechniques, as are outlined herein.

For example, a preferred method to connect a first multiplicity ofcircuits or lines on the chip to a smaller plurality of lines at aconnector leading from the chip are to use a switching device such as amultiplexer (MUX) or relays to selectively couple circuits on the chipor board with circuits off the board.

The number of multiplexers will depend on the number of electrodes inthe array. In one embodiment, a single MUX is utilized. In a preferredembodiment, a plurality of MUXs are used. This can be done in a varietyof ways, as will be appreciated by those in the art; in one embodiment,“sectors” of electrodes are assigned to a particular MUX; thus forexample, rows or columns of the array may each have their own MUX.Alternatively, submultiplexers are used; for example, a column or row isconnected to a respective sub-multiplexer, with the sub-multiplexeroutputs going to another submultiplexer.

In a preferred embodiment, the multiplexer includes a binary counterwhich receives the control signal via the connector pad. The controlsignal is preferably a pulsed signal such as a clock signal andgenerates a sequential count to drive the decoders.

In a preferred embodiment, another way to connect a multiplicity ofelectrodes on the substrate to a smaller number of connector padsleading “off chip” is to use row-column select signals to allow theselection of individual electrodes.

Unfortunately, for structures and methods that access differentelectrodes or groups of electrodes in a time sequential manner, somecorrection or adjustment of the sensed results may be required when thedifference in time is sufficiently large to alter the results, in orderto maintain a calibration between earlier sensed and read-out data andlater sensed and read-out data. The need for such adjustment will dependupon the assay, reaction kinetics and the time separation which may alsobe a function of the number of electrodes to be sensed or read-out. Forexample, in some embodiments it may be entirely reasonable to measureeach of the 25 electrodes in a 5H 5 array of electrodes a few secondsapart (e.g. 10 seconds apart); however, in other embodiments, the 4minute separation between the first and last measurement may beunacceptable or difficult to compensate.

It is also desirable to consider the kinetics of reaction when thereaction takes place on or near a planar surface, such as the surface ofthe electrode. Diffusion rates may play a more important role than whenthe reaction occurs in solution. It is important to understand when orover what period of time the reaction takes place so that themeasurements are taken at the appropriate time. This may be particularlyimportant if an intermediate reaction product is to be sensed, or if aseries of measurements are desirable, for example to do reactionkinetics.

Reaction kinetics are also an important consideration for the drivingsignals. Biosensors are limited by the chemical kinetics. For the classof molecules of interest here (DNA, DNA fragments, proteins, antibodies,and the like), each molecule has a maximum speed or velocity in themedium. For example, the molecules may typically be actively driven ormoved in solution at frequencies between about 1 Hz and 10 kHz, moretypically between about 5 Hz and 5 kHz. At higher frequencies, themolecules only vibrate, while at lower frequencies the movement is notparticularly useful.

In addition, there is an assay volume, that is the accessible volume ofthe assay, associated with each driving signal frequency. As thefrequency increases, the assay volume shrinks in size and volume. Thishas implication for the distribution of electrodes and the drivingsignal frequency.

One additional consideration for sensing or measuring a reaction is thepossible effect that the reaction medium (such as solution components,sample components, reaction components, etc.) may have on theelectrodes. Sometimes the electrodes will degrade, become passivated, orotherwise change over time thereby affecting the accuracy and uniformityof measurements. Under such conditions it is desirable to perform thesensing, measurement, or analysis quickly, or at least according topredetermined timings so that the data collected will be properlyinterpreted.

In a preferred embodiment, one or more preamplifiers are used. As willbe appreciated by those in the art, the preamplifier can be on thesurface of the substrate, e.g. “on board” or “on chip”, or may beprovided in circuitry external to the array chip. It is preferable,however, that the preamplifier be included on the substrate to increasethe signal-to-noise ratio of the signal provided to the externalcircuitry.

In a preferred embodiment, each individual electrode has an associatedpreamplifier.

In a preferred embodiment, the array is divided into “sectors”, whereina subset of the electrodes in the array have an associated MUX andpreamplifier. Similarly, other components of the invention may beassociated with sectors.

In a preferred embodiment, impedance matching is done.

In a preferred embodiment, filters are used, including, but not limitedto, time domain filters and frequency domain filters, and combinations.

In addition to electronic components, the electrodes of the invention inpreferred embodiments comprise self-assembled monolayers (SAMs). Thecompositions of these SAMs will vary with the detection method used. Ingeneral, there are two basic detection mechanisms. In a preferredembodiment, detection of an ETM is based on electron transfer throughthe stacked n-orbitals of double stranded nucleic acid. This basicmechanism is described in U.S. Pat. Nos. 5,591,578, 5,770,369,5,705,348, and PCT US97/20014 and is termed “mechanism-1” herein.Briefly, previous work has shown that electron transfer can proceedrapidly through the stacked n-orbitals of double stranded nucleic acid,and significantly more slowly through single-stranded nucleic acid.Accordingly, this can serve as the basis of an assay. Thus, by addingETMs (either covalently to one of the strands or non-covalently to thehybridization complex through the use of hybridization indicators,described below) to a nucleic acid that is attached to a detectionelectrode via a conductive oligomer, electron transfer between the ETMand the electrode, through the nucleic acid and conductive oligomer, maybe detected.

Alternatively, the ETM can be detected, not necessarily via electrontransfer through nucleic acid, but rather can be directly detected on anelectrode comprising a SAM; that is, the electrons from the ETMs neednot travel through the stacked n orbitals in order to generate a signal.As above, in this embodiment, the detection electrode preferablycomprises a self-assembled monolayer (SAM) that serves to shield theelectrode from redox-active species in the sample. In this embodiment,the presence of ETMs on the surface of a SAM, that has been formulatedto comprise slight “defects” (sometimes referred to herein as“microconduits”, “nanoconduits” or “electroconduits”) can be directlydetected. This basic idea is termed “mechanism-2” herein. Essentially,the electroconduits allow particular ETMs access to the surface. Withoutbeing bound by theory, it should be noted that the configuration of theelectroconduit depends in part on the ETM chosen. For example, the useof relatively hydrophobic ETMs allows the use of hydrophobicelectroconduit forming species, which effectively exclude hydrophilic orcharged ETMs. Similarly, the use of more hydrophilic or charged speciesin the SAM may serve to exclude hydrophobic ETMs.

It should be noted that these defects are to be distinguished from“holes” that allow direct contact of sample components with thedetection electrode. As is more fully outlined below, theelectroconduits can be generated in several general ways, including butnot limited to the use of rough electrode surfaces, such as goldelectrodes formulated on PC circuit boards; or the inclusion of at leasttwo different species in the monolayer, i.e. using a “mixed monolayer”,at least one of which is a electroconduit-forming species (EFS). Thus,upon binding of a target analyte, a soluble binding ligand comprising anETM is brought to the surface, and detection of the ETM can proceed,putatively through the “electroconduits” to the electrode. Essentially,the role of the SAM comprising the defects is to allow electroniccontact of the ETM with the electronic surface of the electrode, whilestill providing the benefits of shielding the electrode from solutioncomponents and reducing the amount of non-specific binding to theelectrodes. Viewed differently, the role of the binding ligand is toprovide specificity for a recruitment of ETMs to the surface, where theycan be directly detected.

Thus, in either embodiment, as is more fully outlined below, an assaycomplex is formed that contains an ETM, which is then detected using thedetection electrode.

Thus, in a preferred embodiment, the electrode comprises a monolayer,comprising electroconduit forming species (EFS). As outlined herein, theefficiency of target analyte binding (for example, oligonucleotidehybridization) may increase when the analyte is at a distance from theelectrode. Similarly, non-specific binding of biomolecules, includingthe target analytes, to an electrode is generally reduced when amonolayer is present. Thus, a monolayer facilitates the maintenance ofthe analyte away from the electrode surface. In addition, a monolayerserves to keep charged species away from the surface of the electrode.Thus, this layer helps to prevent electrical contact between theelectrodes and the ETMs, or between the electrode and charged specieswithin the solvent. Such contact can result in a direct “short circuit”or an indirect short circuit via charged species which may be present inthe sample. Accordingly, the monolayer is preferably tightly packed in auniform layer on the electrode surface, such that a minimum of “holes”exist. The monolayer thus serves as a physical barrier to block solventaccesibility to the electrode.

By “monolayer” or “self-assembled monolayer” or “SAM” herein is meant arelatively ordered assembly of molecules spontaneously chemisorbed on asurface, in which the molecules are oriented approximately parallel toeach other and roughly perpendicular to the surface. A majority of themolecules include a functional group that adheres to the surface, and aportion that interacts with neighboring molecules in the monolayer toform the relatively ordered array. A “mixed” monolayer comprises aheterogeneous monolayer, that is, where at least two different moleculesmake up the monolayer.

In general, the SAMs of the invention can be generated in a number ofways and comprise a number of different components, depending on theelectrode surface and the system used. For “mechanism-1” embodiments,preferred embodiments utilize two monolayer forming species: a monolayerforming species (including insulators or conductive oligomers) and aconductive oligomer species comprising the capture binding ligand,although as will be appreciated by those in the art, additionalmonolayer forming species can be included as well. For “mechanism-2”systems, the composition of the SAM depends on the detection electrodesurface. In general, two basic “mechanism-2” systems are described;detection electrodes comprising “smooth” surfaces, such as gold ballelectrodes, and those comprising “rough” surfaces, such as those thatare made using commercial processes on PC circuit boards. In general,without being bound by theory, it appears that monolayers made onimperfect surfaces, i.e. “rough” surfaces, spontaneously form monolayerscontaining enough electroconduits even in the absence of EFS, probablydue to the fact that the formation of a uniform monolayer on a roughsurface is difficult. “Smoother” surfaces, however, may require theinclusion of sufficient numbers of EFS to generate the electroconduits,as the uniform surfaces allow a more uniform monolayer to form. Again,without being bound by theory, the inclusion of species that disturb theuniformity of the monolayer, for example by including a rigid moleculein a background of more flexible ones, causes electroconduits. Thus“smooth” surfaces comprise monolayers comprising three components: aninsulator species, a EFS, and a species comprising the capture ligand,although in some circumstances, for example when the capture ligandspecies is included at high density, the capture ligand species canserve as the EFS. “Smoothness” in this context is not measuredphysically but rather as a function of an increase in the measuredsignal when EFS are included. That is, the signal from a detectionelectrode coated with monolayer forming species is compared to a signalfrom a detection electrode coated with monolayer forming speciesincluding a EFS. An increase indicates that the surface is relativelysmooth, since the inclusion of a EFS served to facilitate the access ofthe ETM to the electrode. It should also be noted that while thediscussion herein is mainly directed to gold electrodes andthiol-containing monolayer forming species, other types of electrodesand monolayer-forming species can be used.

It should be noted that the “electroconduits” of mechanism-2 systems donot result in direct contact of sample components with the electrodesurface; that is, the electroconduits are not large pores or holes thatallow physical access to the electrode. Rather, without being bound bytheory, it appears that the electroconduits allow certain types of ETMs,particularly hydrophobic ETMs, to penetrate sufficiently into themonolayer to allow detection. However, other types of redox activespecies, including some hydrophilic species, do not penetrate into themonolayer, even with electroconduits present. Thus, in general, redoxactive species that may be present in the sample do not give substantialsignals as a result of the electroconduits. While the exact system willvary with the composition of the SAM and the choice of the ETM, ingeneral, the test for a suitable SAM to reduce non-specific binding thatalso has sufficient electroconduits for ETM detection is to add eitherferrocene or ferrocyanide to the SAM; the former should give a signaland the latter should not.

Accordingly, in mechanism-1 systems, the monolayer comprises a firstspecies comprising a conductive oligomer comprising the capture bindingligand, as is more fully outlined below, and a second species comprisinga monolayer forming species, including either or both insulators orconductive oligomers.

In a preferred embodiment, the monolayer compriseselectroconduit-forming species. By “electroconduit-forming species” or“EFS” herein is meant a molecule that is capable of generatingsufficient electroconduits in a monolayer, generally of insulators suchas alkyl groups, to allow detection of ETMs at the surface. In general,EFSs have one or more of the following qualities: they may be relativelyrigid molecules, for example as compared to an alkyl chain; they mayattach to the electrode surface with a geometry different from the othermonolayer forming species (for example, alkyl chains attached to goldsurfaces with thiol groups are thought to attach at roughly 45° angles,and phenyl-acetylene chains attached to gold via thiols are thought togo down at 90° angles); they may have a structure that stericallyinterferes or interrupts the formation of a tightly packed monolayer,for example through the inclusion of branching groups such as alkylgroups, or the inclusion of highly flexible species, such aspolyethylene glycol units; or they may be capable of being activated toform electroconduits; for example, photoactivatible species that can beselectively removed from the surface upon photoactivation, leavingelectroconduits.

Preferred EFSs include conductive oligomers, as defined below, andphenyl-acetylene-polyethylene glycol species, as well as asymmetricalSAM-forming disulfide species such as described in U.S. Ser. No.09/847,113, filed May 1, 2001, hereby expressly incorporated byreference. However, in some embodiments, the EFS is not a conductiveoligomer.

In a preferred embodiment, the monolayer comprises conductive oligomers.By “conductive oligomer” herein is meant a substantially conductingoligomer, preferably linear, some embodiments of which are referred toin the literature as “molecular wires”. By “substantially conducting”herein is meant that the oligomer is capable of transferring electronsat 100 Hz. Generally, the conductive oligomer has substantiallyoverlapping n-orbitals, i.e. conjugated n-orbitals, as between themonomeric units of the conductive oligomer, although the conductiveoligomer may also contain one or more sigma (σ) bonds. Additionally, aconductive oligomer may be defined functionally by its ability to injector receive electrons into or from an associated ETM. Furthermore, theconductive oligomer is more conductive than the insulators as definedherein. Additionally, the conductive oligomers of the invention are tobe distinguished from electroactive polymers, that themselves may donateor accept electrons.

In a preferred embodiment, the conductive oligomers have a conductivity,S, of from between about 10⁻⁶ to about 10⁴ Ω⁻¹cm⁻¹, with from about 10⁻⁵to about 10³ Ω⁻¹cm⁻¹ being preferred, with these S values beingcalculated for molecules ranging from about 20Δ to about 200Δ. Asdescribed below, insulators have a conductivity S of about 10⁻⁷ Ω⁻¹cm⁻¹or lower, with less than about 10⁻⁸ Ω⁻¹ cm⁻¹ being preferred. Seegenerally Gardner et al., Sensors and Actuators A 51 (1995) 57-66,incorporated herein by reference.

Desired characteristics of a conductive oligomer include highconductivity, sufficient solubility in organic solvents and/or water forsynthesis and use of the compositions of the invention, and preferablychemical resistance to reactions that occur i) during nucleic acidsynthesis (such that nucleosides containing the conductive oligomers maybe added to a nucleic acid synthesizer during the synthesis of thecompositions of the invention), ii) during the attachment of theconductive oligomer to an electrode, or iii) during hybridizationassays. In addition, conductive oligomers that will promote theformation of self-assembled monolayers are preferred.

The oligomers of the invention comprise at least two monomeric subunits,as described herein. As is described more fully below, oligomers includehomo- and hetero-oligomers, and include polymers.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 1:

As will be understood by those in the art, all of the structuresdepicted herein may have additional atoms or structures; e.g. theconductive oligomer of Structure 1 may be attached to ETMs, such aselectrodes, transition metal complexes, organic ETMs, and metallocenes,and to nucleic acids, or to several of these. Unless otherwise noted,the conductive oligomers depicted herein will be attached at the leftside to an electrode; that is, as depicted in Structure 1, the left “Y”is connected to the electrode as described herein. If the conductiveoligomer is to be attached to a nucleic acid, the right “Y”, if present,is attached to the nucleic acid, either directly or through the use of alinker, as is described herein.

In this embodiment, Y is an aromatic group, n is an integer from 1 to50, g is either 1 or zero, e is an integer from zero to 10, and m iszero or 1. When g is 1, B-D is a bond able to conjugate with neighboringbonds (herein referred to as a “conjugated bond”), preferably selectedfrom acetylene, B-D is a conjugated bond, preferably selected fromacetylene, alkene, substituted alkene, amide, azo, —C═N— (including—N═C—, —CR═N— and —N═CR—), —Si═Si—, and —Si═C— (including —C═Si—,—Si═CR— and —CR═Si—). When g is zero, e is preferably 1, D is preferablycarbonyl, or a heteroatom moiety, wherein the heteroatom is selectedfrom oxygen, sulfur, nitrogen, silicon or phosphorus. Thus, suitableheteroatom moieties include, but are not limited to, —NH and —NR,wherein R is as defined herein; substituted sulfur; sulfonyl (—SO₂—)sulfoxide (—SO—); phosphine oxide (—PO— and —RPO—); and thiophosphine(—PS— and —RPS—). However, when the conductive oligomer is to beattached to a gold electrode, as outlined below, sulfur derivatives arenot preferred.

By “aromatic group” or grammatical equivalents herein is meant anaromatic monocyclic or polycyclic hydrocarbon moiety generallycontaining 5 to 14 carbon atoms (although larger polycyclic ringsstructures may be made) and any carbocylic ketone or thioketonederivative thereof, wherein the carbon atom with the free valence is amember of an aromatic ring. Aromatic groups include arylene groups andaromatic groups with more than two atoms removed. For the purposes ofthis application aromatic includes heterocycle. “Heterocycle” or“heteroaryl” means an aromatic group wherein 1 to 5 of the indicatedcarbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen,sulfur, phosphorus, boron and silicon wherein the atom with the freevalence is a member of an aromatic ring, and any heterocyclic ketone andthioketone derivative thereof. Thus, heterocycle includes thienyl,furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,isoquinolyl, thiazolyl, imidozyl, etc.

Importantly, the Y aromatic groups of the conductive oligomer may bedifferent, i.e. the conductive oligomer may be a heterooligomer. Thatis, a conductive oligomer may comprise a oligomer of a single type of Ygroups, or of multiple types of Y groups.

The aromatic group may be substituted with a substitution group,generally depicted herein as R. R groups may be added as necessary toaffect the packing of the conductive oligomers, i.e. R groups may beused to alter the association of the oligomers in the monolayer. Rgroups may also be added to 1) alter the solubility of the oligomer orof compositions containing the oligomers; 2) alter the conjugation orelectrochemical potential of the system; and 3) alter the charge orcharacteristics at the surface of the monolayer.

In a preferred embodiment, when the conductive oligomer is greater thanthree subunits, R groups are preferred to increase solubility whensolution synthesis is done. However, the R groups, and their positions,are chosen to minimally effect the packing of the conductive oligomerson a surface, particularly within a monolayer, as described below. Ingeneral, only small R groups are used within the monolayer, with largerR groups generally above the surface of the monolayer. Thus for examplethe attachment of methyl groups to the portion of the conductiveoligomer within the monolayer to increase solubility is preferred, withattachment of longer alkoxy groups, for example, C3 to C10, ispreferably done above the monolayer surface. In general, for the systemsdescribed herein, this generally means that attachment of stericallysignificant R groups is not done on any of the first two or threeoligomer subunits, depending on the average length of the moleculesmaking up the monolayer.

Suitable R groups include, but are not limited to, hydrogen, alkyl,alcohol, aromatic, amino, amido, nitro, ethers, esters, aldehydes,sulfonyl, silicon moieties, halogens, sulfur containing moieties,phosphorus containing moieties, and ethylene glycols. In the structuresdepicted herein, R is hydrogen when the position is unsubstituted. Itshould be noted that some positions may allow two substitution groups, Rand R′, in which case the R and R′ groups may be either the same ordifferent.

By “alkyl group” or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. The alkyl group may range from about1 to about 30 carbon atoms (C1-C30), with a preferred embodimentutilizing from about 1 to about 20 carbon atoms (C1-C20), with about C1through about C12 to about C15 being preferred, and C1 to C5 beingparticularly preferred, although in some embodiments the alkyl group maybe much larger. Also included within the definition of an alkyl groupare cycloalkyl groups such as C5 and C6 rings, and heterocyclic ringswith nitrogen, oxygen, sulfur or phosphorus. Alkyl also includesheteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and siliconebeing preferred. Alkyl includes substituted alkyl groups. By“substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above.

By “amino groups” or grammatical equivalents herein is meant —NH₂, —NHRand —NR₂ groups, with R being as defined herein.

By “nitro group” herein is meant an —NO₂ group.

By “sulfur containing moieties” herein is meant compounds containingsulfur atoms, including but not limited to, thia-, thio- andsulfo-compounds, thiols (—SH and —SR), and sulfides (—RSR—). By“phosphorus containing moieties” herein is meant compounds containingphosphorus, including, but not limited to, phosphines and phosphates. By“silicon containing moieties” herein is meant compounds containingsilicon.

By “ether” herein is meant an —O—R group. Preferred ethers includealkoxy groups, with —O—(CH₂)₂CH₃ and —O—(CH₂)₄—CH₃ being preferred.

By “ester” herein is meant a —COOR group.

By “halogen” herein is meant bromine, iodine, chlorine, or fluorine.Preferred substituted alkyls are partially or fully halogenated alkylssuch as CF₃, etc.

By “aldehyde” herein is meant —RCHO groups.

By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.

By “amido” herein is meant —RCONH— or RCONR— groups.

By “ethylene glycol” or “(poly)ethylene glycol” herein is meant a—(O—CH₂—CH₂)_(n)— group, although each carbon atom of the ethylene groupmay also be singly or doubly substituted, e.g.—(O—CR₂—CR₂)_(n)—, with Ras described above. Ethylene glycol derivatives with other heteroatomsin place of oxygen (i.e.—(N—CH₂—CH₂)_(n)— or —(S—CH₂—CH₂)_(n)—, or withsubstitution groups) are also preferred.

Preferred substitution groups include, but are not limited to, methyl,ethyl, propyl, alkoxy groups such as —O—(CH₂)₂CH₃ and —O—(CH₂)₄—CH₃ andethylene glycol and derivatives thereof.

Preferred aromatic groups include, but are not limited to, phenyl,naphthyl, naphthalene, anthracene, phenanthroline, pyrole, pyridine,thiophene, porphyrins, and substituted derivatives of each of these,included fused ring derivatives.

In the conductive oligomers depicted herein, when g is 1, B-D is a bondlinking two atoms or chemical moieties. In a preferred embodiment, B-Dis a conjugated bond, containing overlapping or conjugated n-orbitals.

Preferred B-D bonds are selected from acetylene (—C≡C—, also calledalkyne or ethyne), alkene (—CH═CH—, also called ethylene), substitutedalkene (—CR═CR—, —CH═CR— and —CR═CH—), amide (—NH—CO— and —NR—CO— or—CO—NH— and —CO—NR—), azo (—N═N—), esters and thioesters (—CO—O—,—O—CO—, —CS—O— and —O—CS—) and other conjugated bonds such as (—CH═N—,—CR═N—, —N═CH— and —N═CR—), (—SiH═SiH—, —SiR═SiH—, —SiR═SiH—, and—SiR═SiR—), (—SiH═CH—, —SiR═CH—, —SiH═CR—, —SiR═CR—, —CH═SiH—, —CR═SiH—,—CH═SiR—, and —CR═SiR—). Particularly preferred B-D bonds are acetylene,alkene, amide, and substituted derivatives of these three, and azo.Especially preferred B-D bonds are acetylene, alkene and amide. Theoligomer components attached to double bonds may be in the trans or cisconformation, or mixtures. Thus, either B or D may include carbon,nitrogen or silicon. The substitution groups are as defined as above forR.

When g=0 in the Structure 1 conductive oligomer, e is preferably 1 andthe D moiety may be carbonyl or a heteroatom moiety as defined above.

As above for the Y rings, within any single conductive oligomer, the B-Dbonds (or D moieties, when g=0) may be all the same, or at least one maybe different. For example, when m is zero, the terminal B-D bond may bean amide bond, and the rest of the B-D bonds may be acetylene bonds.Generally, when amide bonds are present, as few amide bonds as possibleare preferable, but in some embodiments all the B-D bonds are amidebonds. Thus, as outlined above for the Y rings, one type of B-D bond maybe present in the conductive oligomer within a monolayer as describedbelow, and another type above the monolayer level, for example to givegreater flexibility for nucleic acid hybridization when the nucleic acidis attached via a conductive oligomer.

In the structures depicted herein, n is an integer from 1 to 50,although longer oligomers may also be used (see for example Schumm etal., Angew. Chem. Int. Ed. Engl. 1994 33(13):1360). Without being boundby theory, it appears that for efficient hybridization of nucleic acidson a surface, the hybridization should occur at a distance from thesurface, i.e. the kinetics of hybridization increase as a function ofthe distance from the surface, particularly for long oligonucleotides of200 to 300 basepairs. Accordingly, when a nucleic acid is attached via aconductive oligomer, as is more fully described below, the length of theconductive oligomer is such that the closest nucleotide of the nucleicacid is positioned from about 6Δ to about 100Δ (although distances of upto 500Δ, may be used) from the electrode surface, with from about 15Δ toabout 60Δ being preferred and from about 25Δ to about 60Δ also beingpreferred. Accordingly, n will depend on the size of the aromatic group,but generally will be from about 1 to about 20, with from about 2 toabout 15 being preferred and from about 3 to about 10 being especiallypreferred.

In the structures depicted herein, m is either 0 or 1. That is, when mis 0, the conductive oligomer may terminate in the B-D bond or D moiety,i.e. the D atom is attached to the nucleic acid either directly or via alinker. In some embodiments, for example when the conductive oligomer isattached to a phosphate of the ribose-phosphate backbone of a nucleicacid, there may be additional atoms, such as a linker, attached betweenthe conductive oligomer and the nucleic acid. Additionally, as outlinedbelow, the D atom may be the nitrogen atom of the amino-modified ribose.Alternatively, when m is 1, the conductive oligomer may terminate in Y,an aromatic group, i.e. the aromatic group is attached to the nucleicacid or linker.

As will be appreciated by those in the art, a large number of possibleconductive oligomers may be utilized. These include conductive oligomersfalling within the Structure 1 and Structure 8 formulas, as well asother conductive oligomers, as are generally known in the art, includingfor example, compounds comprising fused aromatic rings or Teflon®-likeoligomers, such as —(CF₂)_(n)—, —(CHF)_(n)— and —(CFR)_(n)—. See forexample, Schumm et al., Angew. Chem. Intl. Ed. Engl. 33:1361 (1994);Grosshenny et al., Platinum Metals Rev. 40(1):26-35 (1996); Tour, Chem.Rev. 96:537-553 (1996); Hsung et al., Organometallics 14:4808-4815(1995); and references cited therein, all of which are expresslyincorporated by reference.

Particularly preferred conductive oligomers of this embodiment aredepicted below:

Structure 2 is Structure 1 when g is 1. Preferred embodiments ofStructure 2 include: e is zero, Y is pyrrole or substituted pyrrole; eis zero, Y is thiophene or substituted thiophene; e is zero, Y is furanor substituted furan; e is zero, Y is phenyl or substituted phenyl; e iszero, Y is pyridine or substituted pyridine; e is 1, B-D is acetyleneand Y is phenyl or substituted phenyl (see Structure 4 below). Apreferred embodiment of Structure 2 is also when e is one, depicted asStructure 3 below:

Preferred embodiments of Structure 3 are: Y is phenyl or substitutedphenyl and B-D is azo; Y is phenyl or substituted phenyl and B-D isacetylene; Y is phenyl or substituted phenyl and B-D is alkene; Y ispyridine or substituted pyridine and B-D is acetylene; Y is thiophene orsubstituted thiophene and B-D is acetylene; Y is furan or substitutedfuran and B-D is acetylene; Y is thiophene or furan (or substitutedthiophene or furan) and B-D are alternating alkene and acetylene bonds.

Most of the structures depicted herein utilize a Structure 3 conductiveoligomer. However, any Structure 3 oligomers may be substituted with anyof the other structures depicted herein, i.e. Structure 1 or 8 oligomer,or other conducting oligomer, and the use of such Structure 3 depictionis not meant to limit the scope of the invention.

Particularly preferred embodiments of Structure 3 include Structures 4,5, 6 and 7, depicted below:

Particularly preferred embodiments of Structure 4 include: n is two, mis one, and R is hydrogen; n is three, m is zero, and R is hydrogen; andthe use of R groups to increase solubility.

When the B-D bond is an amide bond, as in Structure 5, the conductiveoligomers are pseudopeptide oligomers. Although the amide bond inStructure 5 is depicted with the carbonyl to the left, i.e. —CONH—, thereverse may also be used, i.e. —NHCO—. Particularly preferredembodiments of Structure 5 include: n is two, m is one, and R ishydrogen; n is three, m is zero, and R is hydrogen (in this embodiment,the terminal nitrogen (the D atom) may be the nitrogen of theamino-modified ribose); and the use of R groups to increase solubility.

Preferred embodiments of Structure 6 include the first n is two, secondn is one, m is zero, and all R groups are hydrogen, or the use of Rgroups to increase solubility.

Preferred embodiments of Structure 7 include: the first n is three, thesecond n is from 1-3, with m being either 0 or 1, and the use of Rgroups to increase solubility.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 8:

In this embodiment, C are carbon atoms, n is an integer from 1 to 50, mis 0 or 1, J is a heteroatom selected from the group consisting ofoxygen, nitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide,and G is a bond selected from alkane, alkene or acetylene, such thattogether with the two carbon atoms the C-G-C group is an alkene(—CH═CH—), substituted alkene (—CR═CR—) or mixtures thereof (—CH═CR— or—CR═CH—), acetylene (—C≡C—), or alkane (—CR₂—CR₂—, with R being eitherhydrogen or a substitution group as described herein). The G bond ofeach subunit may be the same or different than the G bonds of othersubunits; that is, alternating oligomers of alkene and acetylene bondscould be used, etc. However, when G is an alkane bond, the number ofalkane bonds in the oligomer should be kept to a minimum, with about sixor less sigma bonds per conductive oligomer being preferred. Alkenebonds are preferred, and are generally depicted herein, although alkaneand acetylene bonds may be substituted in any structure or embodimentdescribed herein as will be appreciated by those in the art.

In some embodiments, for example when ETMs are not present, if m=0 thenat least one of the G bonds is not an alkane bond.

In a preferred embodiment, the m of Structure 8 is zero. In aparticularly preferred embodiment, m is zero and G is an alkene bond, asis depicted in Structure 9:

The alkene oligomer of structure 9, and others depicted herein, aregenerally depicted in the preferred trans configuration, althougholigomers of cis or mixtures of trans and cis may also be used. Asabove, R groups may be added to alter the packing of the compositions onan electrode, the hydrophilicity or hydrophobicity of the oligomer, andthe flexibility, i.e. the rotational, torsional or longitudinalflexibility of the oligomer. n is as defined above.

In a preferred embodiment, R is hydrogen, although R may be also alkylgroups and polyethylene glycols or derivatives.

In an alternative embodiment, the conductive oligomer may be a mixtureof different types of oligomers, for example of structures 1 and 8.

In addition, in some embodiments, the terminus of at least some of theconductive oligomers in the monolayer are electronically exposed. By“electronically exposed” herein is meant that upon the placement of anETM in close proximity to the terminus, and after initiation with theappropriate signal, a signal dependent on the presence of the ETM may bedetected. The conductive oligomers may or may not have terminal groups.Thus, in a preferred embodiment, there is no additional terminal group,and the conductive oligomer terminates with one of the groups depictedin Structures 1 to 9; for example, a B-D bond such as an acetylene bond.Alternatively, in a preferred embodiment, a terminal group is added,sometimes depicted herein as “Q”. A terminal group may be used forseveral reasons; for example, to contribute to the electronicavailability of the conductive oligomer for detection of ETMs, or toalter the surface of the SAM for other reasons, for example to preventnon-specific binding. For example, there may be negatively chargedgroups on the terminus to form a negatively charged surface such thatwhen the nucleic acid is DNA or RNA the nucleic acid is repelled orprevented from lying down on the surface, to facilitate hybridization.Preferred terminal groups include —NH₂, —OH, —COOH, and alkyl groupssuch as —CH₃, and (poly)alkyloxides such as (poly)ethylene glycol, with—OCH₂CH₂OH, —(OCH₂CH₂O)₂H, —(OCH₂CH₂O)₃H, and —(OCH₂CH₂O)₄H beingpreferred.

In one embodiment, it is possible to use mixtures of conductiveoligomers with different types of terminal groups. Thus, for example,some of the terminal groups may facilitate detection, and some mayprevent non-specific binding.

It will be appreciated that the monolayer may comprise differentconductive oligomer species, although preferably the different speciesare chosen such that a reasonably uniform SAM can be formed. Thus, forexample, when nucleic acids are covalently attached to the electrodeusing conductive oligomers, it is possible to have one type ofconductive oligomer used to attach the nucleic acid, and another typefunctioning to detect the ETM. Similarly, it may be desirable to havemixtures of different lengths of conductive oligomers in the monolayer,to help reduce non-specific signals. Thus, for example, preferredembodiments utilize conductive oligomers that terminate below thesurface of the rest of the monolayer, i.e. below the insulator layer, ifused, or below some fraction of the other conductive oligomers.Similarly, the use of different conductive oligomers may be done tofacilitate monolayer formation, or to make monolayers with alteredproperties.

In a preferred embodiment, the monolayer forming species are“interrupted” conductive oligomers, containing an alkyl portion in themiddle of the conductive oligomer.

In a preferred embodiment, the monolayer comprises photoactivatablespecies as EFSs. This general scheme is depicted in FIG. 11 of Ser. No.09/626,096, incorporated by reference. Photoactivatable species areknown in the art, and include 4,5-dimethoxy-2-nitrobenzyl ester, whichcan be photolyzed at 365 nm for 2 hours.

In a preferred embodiment, the monolayer may further comprise insulatormoieties. By “insulator” herein is meant a substantially nonconductingoligomer, preferably linear. By “substantially nonconducting” herein ismeant that the insulator will not transfer electrons at 100 Hz. The rateof electron transfer through the insulator is preferably slower than therate through the conductive oligomers described herein.

In a preferred embodiment, the insulators have a conductivity, S, ofabout 10⁻⁷ Ω⁻¹ cm⁻¹ or lower, with less than about 10⁻⁸ Ω⁻¹cm⁻¹ beingpreferred. See generally Gardner et al., supra.

Generally, insulators are alkyl or heteroalkyl oligomers or moietieswith sigma bonds, although any particular insulator molecule may containaromatic groups or one or more conjugated bonds. By “heteroalkyl” hereinis meant an alkyl group that has at least one heteroatom, i.e. nitrogen,oxygen, sulfur, phosphorus, silicon or boron included in the chain.Alternatively, the insulator may be quite similar to a conductiveoligomer with the addition of one or more heteroatoms or bonds thatserve to inhibit or slow, preferably substantially, electron transfer.

Suitable insulators are known in the art, and include, but are notlimited to, —(CH₂)_(n)—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethylene glycolor derivatives using other heteroatoms in place of oxygen, i.e. nitrogenor sulfur (sulfur derivatives are not preferred when the electrode isgold).

As for the conductive oligomers, the insulators may be substituted withR groups as defined herein to alter the packing of the moieties orconductive oligomers on an electrode, the hydrophilicity orhydrophobicity of the insulator, and the flexibility, i.e. therotational, torsional or longitudinal flexibility of the insulator. Forexample, branched alkyl groups may be used. Similarly, the insulatorsmay contain terminal groups, as outlined above, particularly toinfluence the surface of the monolayer.

In a preferred embodiment, the insulator species included in the SAMutilizes novel methods and compositions comprising asymmetricdisulfides. As outlined herein, the signals generated from label probescan be dependent on the behavior or properties of the SAM. SAMscomprising “nanoconduits” or “electroconduits”, as outlined in U.S. Ser.No. 60/145,912 hereby expressly incorporated herein by reference in itsentirety, give good signals. Thus, the present invention providesasymmetric insulators based on disulfides, wherein one of the arms beinga longer alkyl chain (or other SAM forming species) and the other armcomprising either a shorter alkyl chain or a bulky group, such as abranched alkyl group, that can be polar or nonpolar) for creating thenanoconduits. Exemplary species and methods of making are described inU.S. Ser. No. 09/847,113. See also Mukaiyama Tetrahedron Lett. 1968,5907; Boustany Tetrahedron Lett. 1970 3547; Harpp Tetrahedron Lett. 19703551; and Oae, J. Chem. Soc. Chem. Commun, 1977, 407, all of which areexpressly incorporated herein by reference.

The length of the species making up the monolayer will vary as needed.As outlined above, it appears that hybridization is more efficient at adistance from the surface. The species to which nucleic acids areattached (as outlined below, these can be either insulators orconductive oligomers) may be basically the same length as the monolayerforming species or longer than them, resulting in the nucleic acidsbeing more accessible to the solvent for hybridization. In someembodiments, the conductive oligomers to which the nucleic acids areattached may be shorter than the monolayer.

As will be appreciated by those in the art, the actual combinations andratios of the different species making up the monolayer can vary widely,and will depend on whether mechanism-1 or -2 is used. Generally, eithertwo or three component systems are preferred for mechanism-2 systems.Three component systems utilize a first species comprising a captureprobe containing species, attached to the electrode via either aninsulator or a conductive oligomer. The second species are conductiveoligomers, and the third species are insulators. In this embodiment, thefirst species can comprise from about 90% to about 1%, with from about20% to about 40% being preferred. For nucleic acids, from about 30% toabout 40% is especially preferred for short oligonucleotide targets andfrom about 10% to about 20% is preferred for longer targets. The secondspecies can comprise from about 1% to about 90%, with from about 20% toabout 90% being preferred, and from about 40% to about 60% beingespecially preferred. The third species can comprise from about 1% toabout 90%, with from about 20% to about 40% being preferred, and fromabout 15% to about 30% being especially preferred. To achieve theseapproximate proportions, preferred ratios of first:second:third speciesin SAM formation solvents are 2:2:1 for short targets, 1:3:1 for longertargets, with total thiol concentration (when used to attach thesespecies, as is more fully outlined below) in the 500 μM to 1 mM range,and 833 μM being preferred.

Alternatively, two component systems can be used. In one embodiment, foruse in either mechanism-1 or mechanism-2 systems, the two components arethe first and second species. In this embodiment, the first species cancomprise from about 1% to about 90%, with from about 1% to about 40%being preferred, and from about 10% to about 40% being especiallypreferred. The second species can comprise from about 1% to about 90%,with from about 10% to about 60% being preferred, and from about 20% toabout 40% being especially preferred. Alternatively, for mechanism-1 ormechanism-2 systems, the two components are the first and the thirdspecies. In this embodiment, the first species can comprise from about1% to about 90%, with from about 1% to about 40% being preferred, andfrom about 10% to about 40% being especially preferred. The secondspecies can comprise from about 1% to about 90%, with from about 10% toabout 60% being preferred, and from about 20% to about 40% beingespecially preferred.

In a preferred embodiment, the deposition of the SAM is done usingaqueous solvents. As is generally described in Steel et al., Anal. Chem.70:4670 (1998), Herne et al., J. Am. Chem. Soc. 119:8916 (1997), andFinklea, Electrochemistry of Organized Monolayers of Thiols and RelatedMolecules on Electrodes, from A. J. Bard, Electroanalytical Chemistry: ASeries of Advances, Vol. 20, Dekker N.Y. 1966-, all of which areexpressly incorporated by reference, the deposition of the SAM-formingspecies can be done out of aqueous solutions, frequently comprisingsalt.

The covalent attachment of the conductive oligomers and insulators maybe accomplished in a variety of ways, depending on the electrode and thecomposition of the insulators and conductive oligomers used. In apreferred embodiment, the attachment linkers with covalently attachednucleosides or nucleic acids as depicted herein are covalently attachedto an electrode. Thus, one end or terminus of the attachment linker isattached to the nucleoside or nucleic acid, and the other is attached toan electrode. In some embodiments it may be desirable to have theattachment linker attached at a position other than a terminus, or evento have a branched attachment linker that is attached to an electrode atone terminus and to two or more nucleosides at other termini, althoughthis is not preferred. Similarly, the attachment linker may be attachedat two sites to the electrode, as is generally depicted in Structures11-13. Generally, some type of linker is used, as depicted below as “A”in Structure 10, where ‘X’ is the conductive oligomer, “I” is aninsulator and the hatched surface is the electrode:

In this embodiment, A is a linker or atom. The choice of “A” will dependin part on the characteristics of the electrode. Thus, for example, Amay be a sulfur moiety when a gold electrode is used. Alternatively,when metal oxide electrodes are used, A may be a silicon (silane) moietyattached to the oxygen of the oxide (see for example Chen et al.,Langmuir 10:3332-3337 (1994); Lenhard et al., J. Electroanal. Chem.78:195-201 (1977), both of which are expressly incorporated byreference). When carbon based electrodes are used, A may be an aminomoiety (preferably a primary amine; see for example Deinhammer et al.,Langmuir 10:1306-1313 (1994)). Thus, preferred A moieties include, butare not limited to, silane moieties, sulfur moieties (including alkylsulfur moieties), and amino moieties. In a preferred embodiment, epoxidetype linkages with redox polymers such as are known in the art are notused.

Although depicted herein as a single moiety, the insulators andconductive oligomers may be attached to the electrode with more than one“A” moiety; the “A” moieties may be the same or different. Thus, forexample, when the electrode is a gold electrode, and “A” is a sulfuratom or moiety, multiple sulfur atoms may be used to attach theconductive oligomer to the electrode, such as is generally depictedbelow in Structures 11, 12 and 13. As will be appreciated by those inthe art, other such structures can be made. In Structures 11, 12 and 13,the A moiety is just a sulfur atom, but substituted sulfur moieties mayalso be used.

It should also be noted that similar to Structure 13, it may be possibleto have a a conductive oligomer terminating in a single carbon atom withthree sulfur moities attached to the electrode. Additionally, althoughnot always depicted herein, the conductive oligomers and insulators mayalso comprise a “Q” terminal group.

In a preferred embodiment, the electrode is a gold electrode, andattachment is via a sulfur linkage as is well known in the art, i.e. theA moiety is a sulfur atom or moiety. Although the exact characteristicsof the gold-sulfur attachment are not known, this linkage is consideredcovalent for the purposes of this invention. A representative structureis depicted in Structure 14, using the Structure 3 conductive oligomer,although as for all the structures depicted herein, any of theconductive oligomers, or combinations of conductive oligomers, may beused. Similarly, any of the conductive oligomers or insulators may alsocomprise terminal groups as described herein. Structure 14 depicts the“A” linker as comprising just a sulfur atom, although additional atomsmay be present (i.e. linkers from the sulfur to the conductive oligomeror substitution groups). In addition, Structure 14 shows the sulfur atomattached to the Y aromatic group, but as will be appreciated by those inthe art, it may be attached to the B-D group (i.e. an acetylene) aswell.

In a preferred embodiment, the electrode is a carbon electrode, i.e. aglassy carbon electrode, and attachment is via a nitrogen of an aminegroup. A representative structure is depicted in Structure 15. Again,additional atoms may be present, i.e. Z type linkers and/or terminalgroups.

In Structure 16, the oxygen atom is from the oxide of the metal oxideelectrode. The Si atom may be combined with other atoms, i.e. be asilicon moiety containing substitution groups. Other attachments forSAMs to other electrodes are known in the art; see for example Napier etal., Langmuir, 1997, for attachment to indium tin oxide electrodes, andalso the chemisorption of phosphates to an indium tin oxide electrode(talk by H. Holden Thorpe, CHI conference, May 4-5, 1998).

The SAMs of the invention can be made in a variety of ways, includingdeposition out of organic solutions and deposition out of aqueoussolutions. The methods outlined herein use a gold electrode as theexample, although as will be appreciated by those in the art, othermetals and methods may be used as well. In one preferred embodiment,indium-tin-oxide (ITO) is used as the electrode.

In a preferred embodiment, a gold surface is first cleaned. A variety ofcleaning procedures may be employed, including, but not limited to,chemical cleaning or etchants including Piranha solution (hydrogenperoxide/sulfuric acid) or aqua regia (hydrochloric acid/nitric acid),electrochemical methods, flame treatment, plasma treatment orcombinations thereof.

Following cleaning, the gold substrate is exposed to the SAM species.When the electrode is ITO, the SAM species are phosphonate-containingspecies. This can also be done in a variety of ways, including, but notlimited to, solution deposition, gas phase deposition, microcontactprinting, spray deposition, deposition using neat components, etc. Apreferred embodiment utilizes a deposition solution comprising a mixtureof various SAM species in solution, generally thiol-containing species.Mixed monolayers that contain nucleic acids are usually prepared using atwo step procedure. The thiolated nucleic acid is deposited during thefirst deposition step (generally in the presence of at least one othermonolayer-forming species) and the mixed monolayer formation iscompleted during the second step in which a second thiol solution minusnucleic acid is added. Optionally, a second step utilizing mild heatingto promote monolayer reorganization.

In a preferred embodiment, the deposition solution is an organicdeposition solution. In this embodiment, a clean gold surface is placedinto a clean vial. A binding ligand deposition solution in organicsolvent is prepared in which the total thiol concentration is betweenmicromolar to saturation; preferred ranges include from about 1 μM to 10mM, with from about 400 uM to about 1.0 mM being especially preferred.In a preferred embodiment, the deposition solution contains thiolmodified DNA (i.e. nucleic acid attached to an attachment linker) andthiol diluent molecules (either conductive oligomers or insulators, withthe latter being preferred). The ratio of nucleic acid to diluent (ifpresent) is usually between 1000:1 to 1:1000, with from about 10:1 toabout 1:10 being preferred and 1:1 being especially preferred. Thepreferred solvents are tetrahydrofuran (THF), acetonitrile,dimethylforamide (DMF), ethanol, or mixtures thereof; generally anysolvent of sufficient polarity to dissolve the capture ligand can beused, as long as the solvent is devoid of functional groups that willreact with the surface. Sufficient nucleic acid deposition solution isadded to the vial so as to completely cover the electrode surface. Thegold substrate is allowed to incubate at ambient temperature or slightlyabove ambient temperature for a period of time ranging from seconds tohours, with 5-30 minutes being preferred. After the initial incubation,the deposition solution is removed and a solution of diluent moleculeonly (from about 1 μM to 10 mM, with from about 100 uM to about 1.0 mMbeing preferred) in organic solvent is added. The gold substrate isallowed to incubate at room temperature or above room temperature for aperiod of time (seconds to days, with from about 10 minutes to about 24hours being preferred). The gold sample is removed from the solution,rinsed in clean solvent and used.

In a preferred embodiment, an aqueous deposition solution is used. Asabove, a clean gold surface is placed into a clean vial. A nucleic aciddeposition solution in water is prepared in which the total thiolconcentration is between about 1 uM and 10 mM, with from about 1 μM toabout 200 uM being preferred. The aqueous solution frequently has saltpresent (up to saturation, with approximately 1 M being preferred),however pure water can be used. The deposition solution contains thiolmodified nucleic acid and often a thiol diluent molecule. The ratio ofnucleic acid to diluent is usually between between 1000:1 to 1:1000,with from about 10:1 to about 1:10 being preferred and 1:1 beingespecially preferred. The nucleic acid deposition solution is added tothe vial in such a volume so as to completely cover the electrodesurface. The gold substrate is allowed to incubate at ambienttemperature or slightly above ambient temperature for 1-30 minutes with5 minutes usually being sufficient. After the initial incubation, thedeposition solution is removed and a solution of diluent molecule-only(10 uM-1.0 mM) in either water or organic solvent is added. The goldsubstrate is allowed to incubate at room temperature or above roomtemperature until a complete monolayer is formed (10 minutes-24 hours).The gold sample is removed from the solution, rinsed in clean solventand used.

In a preferred embodiment, the deposition solution comprises azwitterionic compound, preferably betaine. Preferred embodiments utilizebetaine and Tris-HCl buffers.

In a preferred embodiment, as outlined herein, a circuit board is usedas the substrate for the gold electrodes. Formation of the SAMs on thegold surface is generally done by first cleaning the boards, for examplein a 10% sulfuric acid solution for 30 seconds, detergent solutions,aqua regia, plasma, etc., as outlined herein. Following the sulfuricacid treatment, the boards are washed, for example via immersion in twoMilli-Q water baths for 1 minute each. The boards are then dried, forexample under a stream of nitrogen. Spotting of the deposition solutiononto the boards is done using any number of known spotting systems,generally by placing the boards on an X-Y table, preferably in ahumidity chamber. The size of the spotting drop will vary with the sizeof the electrodes on the boards and the equipment used for delivery ofthe solution; for example, for 250 μM size electrodes, a 30 nanoliterdrop is used. The volume should be sufficient to cover the electrodesurface completely. The drop is incubated at room temperature for aperiod of time (sec to overnight, with 5 minutes preferred) and then thedrop is removed by rinsing in a Milli-Q water bath. The boards are thenoptionally treated with a second deposition solution, generallycomprising insulator in organic solvent, preferably acetonitrile, byimmersion in a 45° C. bath. After 30 minutes, the boards are removed andimmersed in an acetonitrile bath for 30 seconds followed by a milli-Qwater bath for 30 seconds. The boards are dried under a stream ofnitrogen. Preferrably, only the water rinse is employed.

In a preferred embodiment, the detection electrode comprising the SAM(or the sites on the array, for non-electrode embodiments) furthercomprises capture binding ligands, preferably covalently attached. By“binding ligand” or “binding species” herein is meant a compound that isused to probe for the presence of the target analyte, that will bind tothe target analyte. In general, for most of the embodiments describedherein, there are at least two binding ligands used per target analytemolecule; a “capture” or “anchor” binding ligand (also referred toherein as a “capture probe”, particularly in reference to a nucleic acidbinding ligand) that is attached to the detection electrode as describedherein, and a soluble binding ligand (frequently referred to herein as a“signaling probe” or a “label probe”), that binds independently to thetarget analyte, and either directly or indirectly comprises at least oneETM. However, it should be noted that for fluorescence-based nucleicacid detection systems, the target sequence is generally amplified, andduring amplification, a fluorescent label is added; thus these systemsgenerally comprise only two elements, the capture probe and the labeledtarget.

Again, the discussion below is directed to the use of electrodes andelectrochemical detection, but as will be appreciated by those in theart, fluorescent based systems can be used as well.

Generally, the capture binding ligand allows the attachment of a targetanalyte to the detection electrode, for the purposes of detection. As ismore fully outlined below, attachment of the target analyte to thecapture binding ligand may be direct (i.e. the target analyte binds tothe capture binding ligand) or indirect (one or more capture extenderligands may be used).

In a preferred embodiment, the binding is specific, and the bindingligand is part of a binding pair. By “specifically bind” herein is meantthat the ligand binds the analyte, with specificity sufficient todifferentiate between the analyte and other components or contaminantsof the test sample. However, as will be appreciated by those in the art,it will be possible to detect analytes using binding that is not highlyspecific; for example, the systems may use different binding ligands,for example an array of different ligands, and detection of anyparticular analyte is via its “signature” of binding to a panel ofbinding ligands, similar to the manner in which “electronic noses” work.The binding should be sufficient to allow the analyte to remain boundunder the conditions of the assay, including wash steps to removenon-specific binding. In some embodiments, for example in the detectionof certain biomolecules, the binding constants of the analyte to thebinding ligand will be at least about 10⁻⁴ to 10⁻⁹ M⁻¹, with at leastabout 10⁻⁵ to 10⁻⁹ being preferred and at least about 10⁻⁷ to 10⁻⁹ M⁻¹being particularly preferred.

As will be appreciated by those in the art, the composition of thebinding ligand will depend on the composition of the target analyte.Binding ligands to a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is asingle-stranded nucleic acid, the binding ligand is generally asubstantially complementary nucleic acid. Alternatively, as is generallydescribed in U.S. Pat. Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877,5,637,459, 5,683,867, 5,705,337, and related patents, herebyincorporated by reference, nucleic acid “aptamers” can be developed forbinding to virtually any target analyte. Similarly the analyte may be anucleic acid binding protein and the capture binding ligand is either asingle-stranded or double-stranded nucleic acid; alternatively, thebinding ligand may be a nucleic acid binding protein when the analyte isa single or double-stranded nucleic acid. When the analyte is a protein,the binding ligands include proteins (particularly including antibodiesor fragments thereof (FAbs, etc.)), small molecules, or aptamers,described above. Preferred binding ligand proteins include peptides. Forexample, when the analyte is an enzyme, suitable binding ligands includesubstrates, inhibitors, and other proteins that bind the enzyme, i.e.components of a multi-enzyme (or protein) complex. As will beappreciated by those in the art, any two molecules that will associate,preferably specifically, may be used, either as the analyte or thebinding ligand. Suitable analyte/binding ligand pairs include, but arenot limited to, antibodies/antigens, receptors/ligand, proteins/nucleicacids; nucleic acids/nucleic acids, enzymes/substrates and/orinhibitors, carbohydrates (including glycoproteins andglycolipids)/lectins, carbohydrates and other binding partners,proteins/proteins; and protein/small molecules. These may be wild-typeor derivative sequences. In a preferred embodiment, the binding ligandsare portions (particularly the extracellular portions) of cell surfacereceptors that are known to multimerize, such as the growth hormonereceptor, glucose transporters (particularly GLUT4 receptor),transferrin receptor, epidermal growth factor receptor, low densitylipoprotein receptor, high density lipoprotein receptor, leptinreceptor, interleukin receptors including IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15 and IL-17 receptors,VEGF receptor, PDGF receptor, EPO receptor, TPO receptor, ciliaryneurotrophic factor receptor, prolactin receptor, and T-cell receptors.Similarly, there is a wide body of literature relating to thedevelopment of binding partners based on combinatorial chemistrymethods.

In this embodiment, when the binding ligand is a nucleic acid, preferredcompositions and techniques are outlined in U.S. Pat. Nos. 5,591,578;5,824,473; 5,705,348; 5,780,234 and 5,770,369; U.S. Ser. Nos. 08/873,59808/911,589; WO 98/20162; WO98/12430; WO98/57158; WO 00/16089)WO99/57317; WO99/67425; WO00/24941; PCT US00/10903; WO00/38836;WO99/37819; WO99/57319 and PCTUS00/20476; and related materials, all ofwhich are expressly incorporated by reference in their entirety.

The method of attachment of the capture binding ligands to theattachment linker (either an insulator or conductive oligomer) willgenerally be done as is known in the art, and will depend on both thecomposition of the attachment linker and the capture binding ligand. Ingeneral, the capture binding ligands are attached to the attachmentlinker through the use of functional groups on each that can then beused for attachment. Preferred functional groups for attachment areamino groups, carboxy groups, oxo groups and thiol groups. Thesefunctional groups can then be attached, either directly or indirectlythrough the use of a linker, sometimes depicted herein as “Z”. Linkersare well known in the art; for example, homo- or hetero-bifunctionallinkers as are well known (see 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinby reference). Preferred Z linkers include, but are not limited to,alkyl groups (including substituted alkyl groups and alkyl groupscontaining heteroatom moieties), with short alkyl groups, esters, amide,amine, epoxy groups and ethylene glycol and derivatives being preferred,with propyl, acetylene, and C₂ alkene being especially preferred. Z mayalso be a sulfone group, forming sulfonamide linkages.

In this way, capture binding ligands comprising proteins, lectins,nucleic acids, small organic molecules, carbohydrates, etc. can beadded.

A preferred embodiment utilizes proteinaceous capture binding ligands.As is known in the art, any number of techniques may be used to attach aproteinaceous capture binding ligand to an attachment linker. A widevariety of techniques are known to add moieties to proteins.

A preferred embodiment utilizes nucleic acids as the capture bindingligand. While most of the following discussion focuses on nucleic acids,as will be appreciated by those in the art, many of the techniquesoutlined below apply in a similar manner to non-nucleic acid systems aswell, and to systems that rely on attachment to surfaces other thanmetal electrodes.

The capture probe nucleic acid is covalently attached to the electrode,via an “attachment linker”, that can be either a conductive oligomer(required for mechanism-1 systems) or an insulator. By “covalentlyattached” herein is meant that two moieties are attached by at least onebond, including sigma bonds, pi bonds and coordination bonds.

Thus, one end of the attachment linker is attached to a nucleic acid (orother binding ligand), and the other end (although as will beappreciated by those in the art, it need not be the exact terminus foreither) is attached to the electrode. Thus, any of the structuresdepicted herein may further comprise a nucleic acid effectively as aterminal group. Thus, the present invention provides compositionscomprising nucleic acids covalently attached to electrodes as isgenerally depicted below in Structure 17:

In Structure 17, the hatched marks on the left represent an electrode. Xis a conductive oligomer and I is an insulator as defined herein. F₁ isa linkage that allows the covalent attachment of the electrode and theconductive oligomer or insulator, including bonds, atoms or linkers suchas is described herein, for example as “A”, defined below. F₂ is alinkage that allows the covalent attachment of the conductive oligomeror insulator to the nucleic acid, and may be a bond, an atom or alinkage as is herein described. F₂ may be part of the conductiveoligomer, part of the insulator, part of the nucleic acid, or exogeneousto both, for example, as defined herein for “Z”.

In a preferred embodiment, the capture probe nucleic acid is covalentlyattached to the electrode via an attachment linker. The covalentattachment of the nucleic acid and the attachment linker may beaccomplished in several ways. In a preferred embodiment, the attachmentis via attachment to the base of the nucleoside, via attachment to thebackbone of the nucleic acid (either the ribose, the phosphate, or to ananalogous group of a nucleic acid analog backbone), or via a transitionmetal ligand, as described below. The techniques outlined below aregenerally described for naturally occurring nucleic acids, although aswill be appreciated by those in the art, similar techniques may be usedwith nucleic acid analogs, and in some cases with other binding ligands.Similarly, most of the structures herein depict conductive oligomers asthe attachment linkers, but insulators such as alkyl chains arepreferred in many embodiments.

In a preferred embodiment, the attachment linker is attached to the baseof a nucleoside of the nucleic acid. This may be done in several ways,depending on the linker, as is described below. In one embodiment, thelinker is attached to a terminal nucleoside, i.e. either the 3′ or 5′nucleoside of the nucleic acid. Alternatively, the linker is attached toan internal nucleoside.

The point of attachment to the base will vary with the base. Generally,attachment at any position is possible. In some embodiments, for examplewhen the probe containing the ETMs may be used for hybridization (i.e.mechanism-1 systems), it is preferred to attach at positions notinvolved in hydrogen bonding to the complementary base. Thus, forexample, generally attachment is to the 5 or 6 position of pyrimidinessuch as uridine, cytosine and thymine. For purines such as adenine andguanine, the linkage is preferably via the 8 position. Attachment tonon-standard bases is preferably done at the comparable positions.

In one embodiment, the attachment is direct; that is, there are nointervening atoms between the attachment linker and the base. In thisembodiment, for example, attachment linkers comprising conductiveoligomers with terminal acetylene bonds are attached directly to thebase. Structure 18 is an example of this linkage, using a Structure 3conductive oligomer and uridine as the base, although other bases andattachment linkers can be used as will be appreciated by those in theart:

It should be noted that the pentose structures depicted herein may havehydrogen, hydroxy, phosphates or other groups such as amino groupsattached. In addition, the pentose and nucleoside structures depictedherein are depicted non-conventionally, as mirror images of the normalrendering. In addition, the pentose and nucleoside structures may alsocontain additional groups, such as protecting groups, at any position,for example as needed during synthesis.

In addition, the base may contain additional modifications as needed,i.e. the carbonyl or amine groups may be altered or protected.

In an alternative embodiment, the attachment is any number of differentZ linkers, including amide and amine linkages, as is generally depictedin Structure 19 using uridine as the base and a Structure 3 oligomer asthe attachment linker:

In this embodiment, Z is a linker. Preferably, Z is a short linker ofabout 1 to about 10 atoms, with from 1 to 5 atoms being preferred, thatmay or may not contain alkene, alkynyl, amine, amide, azo, imine, etc.,bonds. Linkers are known in the art; for example, homo- orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference).

Preferred Z linkers include, but are not limited to, alkyl groups(including substituted alkyl groups and alkyl groups containingheteroatom moieties), with short alkyl groups, esters, amide, amine,epoxy groups and ethylene glycol and derivatives being preferred, withpropyl, acetylene, and C₂ alkene being especially preferred. Z may alsobe a sulfone group, forming sulfonamide linkages as discussed below.

In a preferred embodiment, the attachment of the nucleic acid and theattachment linker is done via attachment to the backbone of the nucleicacid. This may be done in a number of ways, including attachment to aribose of the ribose-phosphate backbone, or to the phosphate of thebackbone, or other groups of analogous backbones.

As a preliminary matter, it should be understood that the site ofattachment in this embodiment may be to a 3′ or 5′ terminal nucleotide,or to an internal nucleotide, as is more fully described below.

In a preferred embodiment, the attachment linker is attached to theribose of the ribose-phosphate backbone. This may be done in severalways. As is known in the art, nucleosides that are modified at eitherthe 2′ or 3′ position of the ribose with amino groups, sulfur groups,silicone groups, phosphorus groups, or oxo groups can be made (Imazawaet al., J. Org. Chem., 44:2039 (1979); Hobbs et al., J. Org. Chem.42(4):714 (1977); Verheyden et al., J. Orrg. Chem. 36(2):250 (1971);McGee et al., J. Org. Chem. 61:781-785 (1996); Mikhailopulo et al.,Liebigs. Ann. Chem. 513-519 (1993); McGee et al., Nucleosides &Nucleotides 14(6):1329 (1995), all of which are incorporated byreference). These modified nucleosides are then used to add theattachment linkers.

A preferred embodiment utilizes amino-modified nucleosides. Theseamino-modified riboses can then be used to form either amide or aminelinkages to the conductive oligomers. In a preferred embodiment, theamino group is attached directly to the ribose, although as will beappreciated by those in the art, short linkers such as those describedherein for “Z” may be present between the amino group and the ribose.

In a preferred embodiment, an amide linkage is used for attachment tothe ribose. Preferably, if the conductive oligomer of Structures 1-3 isused, m is zero and thus the conductive oligomer terminates in the amidebond. In this embodiment, the nitrogen of the amino group of theamino-modified ribose is the “D” atom of the conductive oligomer. Thus,a preferred attachment of this embodiment is depicted in Structure 20(using the Structure 3 conductive oligomer as the attachment linker):

As will be appreciated by those in the art, Structure 20 has theterminal bond fixed as an amide bond.

In a preferred embodiment, a heteroatom linkage is used, i.e. oxo,amine, sulfur, etc. A preferred embodiment utilizes an amine linkage.Again, as outlined above for the amide linkages, for amine linkages, thenitrogen of the amino-modified ribose may be the “D” atom of theconductive oligomer when the Structure 3 conductive oligomer is used.Thus, for example, Structures 21 and 22 depict nucleosides with theStructures 3 and 9 conductive oligomers, respectively, as the attachmentlinkers, using the nitrogen as the heteroatom, although otherheteroatoms can be used:

In Structure 21, preferably both m and t are not zero. A preferred Zhere is a methylene group, or other aliphatic alkyl linkers. One, two orthree carbons in this position are particularly useful for syntheticreasons.

In Structure 22, Z is as defined above. Suitable linkers includemethylene and ethylene.

In an alternative embodiment, the attachment linker is covalentlyattached to the nucleic acid via the phosphate of the ribose-phosphatebackbone (or analog) of a nucleic acid. In this embodiment, theattachment is direct, utilizes a linker or via an amide bond. Structure23 depicts a direct linkage, and Structure 24 depicts linkage via anamide bond (both utilize the Structure 3 conductive oligomer, althoughStructure 8 conductive oligomers are also possible as well as any numberof other attachment linkers). Structures 23 and 24 depict the conductiveoligomer in the 3′ position, although the 5′ position is also possible.Furthermore, both Structures 23 and 24 depict naturally occurringphosphodiester bonds, although as those in the art will appreciate,non-standard analogs of phosphodiester bonds may also be used.

In Structure 23, if the terminal Y is present (i.e. m=1), thenpreferably Z is not present (i.e. t=0). If the terminal Y is notpresent, then Z is preferably present.

Structure 24 depicts a preferred embodiment, wherein the terminal B-Dbond is an amide bond, the terminal Y is not present, and Z is a linker,as defined herein.

In a preferred embodiment, the attachment linker is covalently attachedto the nucleic acid via a transition metal ligand. In this embodiment,the attachment linker is covalently attached to a ligand which providesone or more of the coordination atoms for a transition metal. In oneembodiment, the ligand to which the attachment linker is attached alsohas the nucleic acid attached, as is generally depicted below inStructure 25. Alternatively, the attachment linker is attached to oneligand, and the nucleic acid is attached to another ligand, as isgenerally depicted below in Structure 26. Thus, in the presence of thetransition metal, the attachment linker is covalently attached to thenucleic acid. Both of these structures depict Structure 3 conductiveoligomers, although other attachment linkers may be utilized. Structures25 and 26 depict two representative structures:

In the structures depicted herein, M is a metal atom, with transitionmetals being preferred. Suitable transition metals for use in theinvention include, but are not limited to, cadmium (Cd), copper (Cu),cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru),rhodium (Rh), osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc),titanium (Ti), Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni),Molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir). Thatis, the first series of transition metals, the platinum metals (Ru, Rh,Pd, Os, Ir and Pt), along with Fe, Re, W, Mo and Tc, are preferred.Particularly preferred are ruthenium, rhenium, osmium, platinum, cobaltand iron.

L are the co-ligands, that provide the coordination atoms for thebinding of the metal ion. As will be appreciated by those in the art,the number and nature of the co-ligands will depend on the coordinationnumber of the metal ion. Mono-, di- or polydentate co-ligands may beused at any position. Thus, for example, when the metal has acoordination number of six, the L from the terminus of the conductiveoligomer, the L contributed from the nucleic acid, and r, add up to six.Thus, when the metal has a coordination number of six, r may range fromzero (when all coordination atoms are provided by the other two ligands)to four, when all the co-ligands are monodentate. Thus generally, r willbe from 0 to 8, depending on the coordination number of the metal ionand the choice of the other ligands.

In one embodiment, the metal ion has a coordination number of six andboth the ligand attached to the conductive oligomer and the ligandattached to the nucleic acid are at least bidentate; that is, r ispreferably zero, one (i.e. the remaining co-ligand is bidentate) or two(two monodentate co-ligands are used).

As will be appreciated in the art, the co-ligands can be the same ordifferent. Suitable ligands fall into two categories: ligands which usenitrogen, oxygen, sulfur, carbon or phosphorus atoms (depending on themetal ion) as the coordination atoms (generally referred to in theliterature as sigma (σ) donors) and organometallic ligands such asmetallocene ligands (generally referred to in the literature as pi (π)donors, and depicted herein as L_(m)). Suitable nitrogen donatingligands are well known in the art and include, but are not limited to,NH₂; NHR; NRR′; pyridine; pyrazine; isonicotinamide; imidazole;bipyridine and substituted derivatives of bipyridine; terpyridine andsubstituted derivatives; phenanthrolines, particularly1,10-phenanthroline (abbreviated phen) and substituted derivatives ofphenanthrolines such as 4,7-dimethylphenanthroline anddipyridol[3,2-a:2′,3′-c]phenazine (abbreviated dppz); dipyridophenazine;1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);9,10-phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam), EDTA, EGTA andisocyanide. Substituted derivatives, including fused derivatives, mayalso be used. In some embodiments, porphyrins and substitutedderivatives of the porphyrin family may be used. See for example,Comprehensive Coordination Chemistry, Ed. Wilkinson et al., PergammonPress, 1987, Chapters 13.2 (pp 73-98), 21.1 (pp. 813-898) and 21.3 (pp915-957), all of which are hereby expressly incorporated by reference.

Suitable sigma donating ligands using carbon, oxygen, sulfur andphosphorus are known in the art. For example, suitable sigma carbondonors are found in Cotton and Wilkenson, Advanced Organic Chemistry,5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference;see page 38, for example. Similarly, suitable oxygen ligands includecrown ethers, water and others known in the art. Phosphines andsubstituted phosphines are also suitable; see page 38 of Cotton andWilkenson.

The oxygen, sulfur, phosphorus and nitrogen-donating ligands areattached in such a manner as to allow the heteroatoms to serve ascoordination atoms.

In a preferred embodiment, organometallic ligands are used. In additionto purely organic compounds for use as redox moieties, and varioustransition metal coordination complexes with σ-bonded organic ligandwith donor atoms as heterocyclic or exocyclic substituents, there isavailable a wide variety of transition metal organometallic compoundswith π-bonded organic ligands (see Advanced Inorganic Chemistry, 5thEd., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26;Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed.,1992, VCH; and Comprehensive Organometallic Chemistry II, A Review ofthe Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10 &11, Pergamon Press, hereby expressly incorporated by reference). Suchorganometallic ligands include cyclic aromatic compounds such as thecyclopentadienide ion [C₅H₅(−1)] and various ring substituted and ringfused derivatives, such as the indenylide (−1) ion, that yield a classof bis(cyclopentadieyl) metal compounds, (i.e. the metallocenes); seefor example Robins et al., J. Am. Chem. Soc. 104:1882-1893 (1982); andGassman et al., J. Am. Chem. Soc. 108:4228-4229 (1986), incorporated byreference. Of these, ferrocene [(C₅H₅)₂Fe] and its derivatives areprototypical examples which have been used in a wide variety of chemical(Connelly et al., Chem. Rev. 96:877-910 (1996), incorporated byreference) and electrochemical (Geiger et al., Advances inOrganometallic Chemistry 23:1-93; and Geiger et al., Advances inOrganometallic Chemistry 24:87, incorporated by reference) electrontransfer or “redox” reactions. Metallocene derivatives of a variety ofthe first, second and third row transition metals are potentialcandidates as redox moieties that are covalently attached to either theribose ring or the nucleoside base of nucleic acid. Other potentiallysuitable organometallic ligands include cyclic arenes such as benzene,to yield bis(arene)metal compounds and their ring substituted and ringfused derivatives, of which bis(benzene)chromium is a prototypicalexample, Other acyclic n-bonded ligands such as the allyl(−1) ion, orbutadiene yield potentially suitable organometallic compounds, and allsuch ligands, in conjuction with other π-bonded and σ-bonded ligandsconstitute the general class of organometallic compounds in which thereis a metal to carbon bond. Electrochemical studies of various dimers andoligomers of such compounds with bridging organic ligands, andadditional non-bridging ligands, as well as with and without metal-metalbonds are potential candidate redox moieties in nucleic acid analysis.

When one or more of the co-ligands is an organometallic ligand, theligand is generally attached via one of the carbon atoms of theorganometallic ligand, although attachment may be via other atoms forheterocyclic ligands. Preferred organometallic ligands includemetallocene ligands, including substituted derivatives and themetalloceneophanes (see page 1174 of Cotton and Wilkenson, supra). Forexample, derivatives of metallocene ligands such asmethylcyclopentadienyl, with multiple methyl groups being preferred,such as pentamethylcyclopentadienyl, can be used to increase thestability of the metallocene. In a preferred embodiment, only one of thetwo metallocene ligands of a metallocene are derivatized.

As described herein, any combination of ligands may be used. Preferredcombinations include: a) all ligands are nitrogen donating ligands; b)all ligands are organometallic ligands; and c) the ligand at theterminus of the attachment linker is a metallocene ligand and the ligandprovided by the nucleic acid is a nitrogen donating ligand, with theother ligands, if needed, are either nitrogen donating ligands ormetallocene ligands, or a mixture. These combinations, using theconductive oligomer of Structure 3, are depicted in Structures 27 (usingphenanthroline and amino as representative ligands), 28 (using ferroceneas the metal-ligand combination) and 29 (using cyclopentadienyl andamino as representative ligands).

Again, other attachment linkers such as alkyl groups may also beutilized.

In a preferred embodiment, the ligands used in the invention showaltered fluorescent properties depending on the redox state of thechelated metal ion. As described below, this thus serves as anadditional mode of detection of electron transfer between the ETM andthe electrode.

In addition, similar methods can be used to attach proteins to thedetection electrode; see for example U.S. Pat. No. 5,620,850, herebyincorporated by reference.

In a preferred embodiment, as is described more fully below, the ligandattached to the nucleic acid is an amino group attached to the 2′ or 3′position of a ribose of the ribose-phosphate backbone. This ligand maycontain a multiplicity of amino groups so as to form a polydentateligand which binds the metal ion. Other preferred ligands includecyclopentadiene and phenanthroline.

In a preferred embodiment, the capture probe nucleic acids (or otherbinding ligands) are covalently attached to the electrode via aninsulator (i.e. the attachment linker is an insulator). The attachmentof nucleic acids (and other binding ligands) to insulators such as alkylgroups is well known, and can be done to the base or the backbone,including the ribose or phosphate for backbones containing thesemoieties, or to alternate backbones for nucleic acid analogs.

In a preferred embodiment, there may be one or more different captureprobe species on the surface. In some embodiments, there may be one typeof capture probe, or one type of capture probe extender, as is morefully described below. Alternatively, different capture probes, or onecapture probe with a multiplicity of different capture extender probescan be used. Similarly, it may be desirable (particularly in the case ofnucleic acid analytes and binding ligands in mechanism-2 systems) to useauxiliary capture probes that comprise relatively short probe sequences,that can be used to “tack down” components of the system, for examplethe recruitment linkers, to increase the concentration of ETMs at thesurface.

In a preferred embodiment, a number of capture probes are designed andused for each target sequence. That is, a single electrode pad of thearray may have 1 probe to the target analyte, or a plurality of probesto the same target sequence, preferably (but not required to be)non-overlapping. This is particularly preferred for long targetsequences. In this embodiment, at least two different capture probes areused, with at least 3, 4, 5, 6, 7, 8, 9 or 10 being preferred, and 8being particularly preferred.

Thus the present invention provides substrates comprising at least onedetection electrode comprising monolayers and capture binding ligands,useful in target analyte detection systems.

In a preferred embodiment, the compositions further comprise a solutionor soluble binding ligand, although as is more fully described below,for mechanism-1 systems, the ETMs may be added in the form ofnon-covalently attached hybridization indicators. Solution bindingligands are similar to capture binding ligands, in that they bind,preferably specifically, to target analytes. The solution binding ligand(generally referred to herein as label probes when the target analytesare nucleic acids) may be the same or different from the capture bindingligand. Generally, the solution binding ligands are not directlyattached to the surface. The solution binding ligand either directlycomprises a recruitment linker that comprises at least one ETM (FIG. 4Afrom 60/190,259), or the recruitment linker binds, either directly (FIG.4A) or indirectly (FIG. 4E), to the solution binding ligand.

Thus, “solution binding ligands” or “soluble binding ligands” or “signalcarriers” or “label probes” or “label binding ligands” with recruitmentlinkers comprising covalently attached ETMs are provided. That is, oneportion of the label probe or solution binding ligand directly orindirectly binds to the target analyte, and one portion comprises arecruitment linker comprising covalently attached ETMs. In some systems,for example in mechanism-1 nucleic acid systems, these may be the same.Similarly, for mechanism-1 systems, the recruitment linker comprisesnucleic acid that will hybridize to detection probes. The terms“electron donor moiety”, “electron acceptor moiety”, and “ETMs” (ETMs)or grammatical equivalents herein refers to molecules capable ofelectron transfer under certain conditions. It is to be understood thatelectron donor and acceptor capabilities are relative; that is, amolecule which can lose an electron under certain experimentalconditions will be able to accept an electron under differentexperimental conditions. It is to be understood that the number ofpossible electron donor moieties and electron acceptor moieties is verylarge, and that one skilled in the art of electron transfer compoundswill be able to utilize a number of compounds in the present invention.Preferred ETMs include, but are not limited to, transition metalcomplexes, organic ETMs, and electrodes.

In a preferred embodiment, the ETMs are transition metal complexes.Transition metals are those whose atoms have a partial or complete dshell of electrons. Suitable transition metals for use in the inventionare listed above.

The transition metals are complexed with a variety of ligands, L,defined above, to form suitable transition metal complexes, as is wellknown in the art.

Preferred ETMs comprise metallocenes, particularly ferrocene.

In addition to transition metal complexes, other organic electron donorsand acceptors may be covalently attached to the nucleic acid for use inthe invention. These organic molecules include, but are not limited to,riboflavin, xanthene dyes, azine dyes, acridine-orange,N,N′-dimethyl-2,7-diazapyrenium dichloride (DAP²⁺), methylviologen,ethidium bromide, quinones such asN,N′-dimethylanthra(2,1,9-def:6,5,10-d′e′f′)diisoquinoline dichloride(ADIQ²⁺); porphyrins ([meso-tetrakis(N-methyl-x-pyridinium)porphyrintetrachloride], varlamine blue B hydrochloride, Bindschedler's green;2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant crestblue (3-amino-9-dimethyl-amino-10-methylphenoxyazine chloride),methylene blue; Nile blue A (aminoaphthodiethylaminophenoxazinesulfate), indigo-5,5′,7,7′-tetrasulfonic acid, indigo-5,5′,7-trisulfonicacid; phenosafranine, indigo-5-monosulfonic acid; safranine T;bis(dimethylglyoximato)-iron(II) chloride; induline scarlet, neutralred, anthracene, coronene, pyrene, 9-phenylanthracene, rubrene,binaphthyl, DPA, phenothiazene, fluoranthene, phenanthrene, chrysene,1,8-diphenyl-1,3,5,7-octatetracene, naphthalene, acenaphthalene,perylene, TMPD and analogs and subsitituted derivatives of thesecompounds.

In one embodiment, the electron donors and acceptors are redox proteinsas are known in the art. However, redox proteins in many embodiments arenot preferred.

The choice of the specific ETMs will be influenced by the type ofelectron transfer detection used, as is generally outlined below.Preferred ETMs are metallocenes, with ferrocene being particularlypreferred.

Accordingly, the present invention provides methods and compositionsuseful in the detection of nucleic acids and other target analytes. Aswill be appreciated by those in the art, the compositions of theinvention can take on a wide variety of configurations. As is more fullyoutlined below, preferred systems of the invention work as follows. Atarget nucleic acid sequence is attached (via hybridization) to anelectrode comprising a monolayer, generally including conductiveoligomers. This attachment can be either directly to a capture probe onthe surface, or indirectly, using capture extender probes. In someembodiments, the target sequence itself comprises the ETMs.Alternatively, a label probe is then added, forming an assay complex.The attachment of the label probe may be direct (i.e. hybridization to aportion of the target sequence), or indirect (i.e. hybridization to anamplifier probe that hybridizes to the target sequence), with all therequired nucleic acids forming an assay complex. As a result of thehybridization of the first portion of the label probe, the secondportion of the label probe, the “recruitment linker”, containing theETMs is brought into spatial proximity to the SAM surface on theelectrode, and the presence of the ETM can then be detectedelectronically. Thus, in a preferred embodiment, the present inventionprovides electrodes comprising monolayers comprising SAM forming speciesand capture probes, useful in nucleic acid (or other target analyte)detection systems. In a preferred embodiment, the compositions furthercomprise a label probe. The label probe is nucleic acid, generallysingle stranded, although as more fully outlined below, it may containdouble-stranded portions. In mechanism-2 systems, the label probecomprises a first portion that is capable of hybridizing to a componentof the assay complex, defined below, and a second portion that does nothybridize to a component of an assay complex and comprises at least onecovalently attached ETM.

Without being bound by theory, it appears that in “mechanism-2” systems,electron transfer is facilitated when the ETM is able to penetrate(“snuggle”) into the monolayer to some degree. That is, in general, itappears that hydrophobic ETMs used with hydrophobic SAMs give rise tobetter (greater) signals than ETMs that are charged or more hydrophilic.Thus, for example, ferrocene in solution can penetrate the monolayers ofthe examples and give a signal when electroconduits are present, whileferrocyanide in solution gives little or no signal. Thus, in general,hydrophobic ETMs are preferred in mechanism-2 systems; however,transition metal complexes, although charged, with one or morehydrophobic ligands, such as Ru and Os complexes, also give rise to goodsignals. Similarly, electron transfer between the ETM and the electrodeis facilitated by the use of linkers or spacers that allow the ETM someflexibility to penetrate into the monolayer; thus the N6 compositions ofthe invention have a four carbon linker attaching the ETM to the nucleicacid.

In a preferred embodiment, a plurality of ETMs are used. The use ofmultiple ETMs provides signal amplification and thus allows moresensitive detection limits. As discussed below, while the use ofmultiple ETMs on nucleic acids that hybridize to complementary strandscan cause decreases in T_(m)s of the hybridization complexes dependingon the number, site of attachment and spacing between the multiple ETMs,this is not a factor when the ETMs are on the recruitment linker, sincethis does not hybridize to a complementary sequence. Accordingly,pluralities of ETMs are preferred, with at least about 2 ETMs perrecruitment linker being preferred, and at least about 10 beingparticularly preferred, and at least about 20 to 50 being especiallypreferred. In some instances, very large numbers of ETMs (100 to 1000)can be used.

As will be appreciated by those in the art, the portion of the labelprobe (or target, in some embodiments) that comprises the ETMs (termedherein a “recruitment linker” or “signal carrier”) can be nucleic acid,or it can be a non-nucleic acid linker that links the first hybridizableportion of the label probe to the ETMs. That is, since this portion ofthe label probe is not required for hybridization, it need not benucleic acid, although this may be done for ease of synthesis. In someembodiments, as is more fully outlined below, the recruitment linker maycomprise double-stranded portions. Thus, as will be appreciated by thosein the art, there are a variety of configurations that can be used. In apreferred embodiment, the recruitment linker is nucleic acid (includinganalogs), and attachment of the ETMs can be via (1) a base; (2) thebackbone, including the ribose, the phosphate, or comparable structuresin nucleic acid analogs; (3) nucleoside replacement, described below; or(4) metallocene polymers, as described below. In a preferred embodiment,the recruitment linker is non-nucleic acid, and can be either ametallocene polymer or an alkyl-type polymer (including heteroalkyl, asis more fully described below) containing ETM substitution groups. Theseoptions are generally depicted in the Figures.

In a preferred embodiment, the recruitment linker is a nucleic acid, andcomprises covalently attached ETMs. The ETMs may be attached tonucleosides within the nucleic acid in a variety of positions. Preferredembodiments include, but are not limited to, (1) attachment to the baseof the nucleoside, (2) attachment of the ETM as a base replacement, (3)attachment to the backbone of the nucleic acid, including either to aribose of the ribose-phosphate backbone or to a phosphate moiety, or toanalogous structures in nucleic acid analogs, and (4) attachment viametallocene polymers, with the latter being preferred.

In addition, as is described below, when the recruitment linker isnucleic acid, it may be desirable to use secondary label probes, thathave a first portion that will hybridize to a portion of the primarylabel probes and a second portion comprising a recruitment linker as isdefined herein. This is generally depicted in FIG. 16H of U.S. Ser. No.60/190,259.

In a preferred embodiment, the ETM is attached to the base of anucleoside as is generally outlined above for attachment of theattachment linkers. Attachment can be to an internal nucleoside or aterminal nucleoside.

The covalent attachment to the base will depend in part on the ETMchosen, but in general is similar to the attachment of conductiveoligomers to bases, as outlined above. Attachment may generally be doneto any position of the base. In a preferred embodiment, the ETM is atransition metal complex, and thus attachment of a suitable metal ligandto the base leads to the covalent attachment of the ETM. Alternatively,similar types of linkages may be used for the attachment of organicETMs, as will be appreciated by those in the art.

In one embodiment, the C4 attached amino group of cytosine, the C6attached amino group of adenine, or the C2 attached amino group ofguanine may be used as a transition metal ligand.

Ligands containing aromatic groups can be attached via acetylenelinkages as is known in the art (see Comprehensive Organic Synthesis,Trost et al., Ed., Pergamon Press, Chapter 2.4: Coupling ReactionsBetween sp² and sp Carbon Centers, Sonogashira, pp 521-549, and pp950-953, hereby incorporated by reference). Structure 30 depicts arepresentative structure in the presence of the metal ion and any othernecessary ligands; Structure 30 depicts uridine, although as for all thestructures herein, any other base may also be used.

L_(a) is a ligand, which may include nitrogen, oxygen, sulfur orphosphorus donating ligands or organometallic ligands such asmetallocene ligands. Suitable L_(a) ligands include, but are not limitedto, phenanthroline, imidazole, bpy and terpy. L_(r) and M are as definedabove. Again, it will be appreciated by those in the art, that a linker(“Z”) may be included between the nucleoside and the ETM.

Similarly, as for the attachment linkers, the linkage may be done usinga linker, which may utilize an amide linkage (see generally Telser etal., J. Am. Chem. Soc. 111:7221-7226 (1989); Telser et al., J. Am. Chem.Soc. 111:7226-7232 (1989), both of which are expressly incorporated byreference). These structures are generally depicted below in Structure31, which again uses uridine as the base, although as above, the otherbases may also be used:

In this embodiment, L is a ligand as defined above, with L_(r) and M asdefined above as well. Preferably, L is amino, phen, byp and terpy.

In a preferred embodiment, the ETM attached to a nucleoside is ametallocene; i.e. the L and L_(r) of Structure 31 are both metalloceneligands, L_(m), as described above. Structure 32 depicts a preferredembodiment wherein the metallocene is ferrocene, and the base isuridine, although other bases may be used:

Preliminary data suggest that Structure 32 may cyclize, with the secondacetylene carbon atom attacking the carbonyl oxygen, forming afuran-like structure. Preferred metallocenes include ferrocene,cobaltocene and osmiumocene.

In a preferred embodiment, the ETM is attached to a ribose at anyposition of the ribose-phosphate backbone of the nucleic acid, i.e.either the 5′ or 3′ terminus or any internal nucleoside. Ribose in thiscase can include ribose analogs. As is known in the art, nucleosidesthat are modified at either the 2′ or 3′ position of the ribose can bemade, with nitrogen, oxygen, sulfur and phosphorus-containingmodifications possible. Amino-modified and oxygen-modified ribose ispreferred. See generally PCT publication WO 95/15971, incorporatedherein by reference. These modification groups may be used as atransition metal ligand, or as a chemically functional moiety forattachment of other transition metal ligands and organometallic ligands,or organic electron donor moieties as will be appreciated by those inthe art. In this embodiment, a linker such as depicted herein for “Z”may be used as well, or a conductive oligomer between the ribose and theETM. Preferred embodiments utilize anachment at the 2′ or 3′ position ofthe ribose, with the 2′ position being preferred. Thus for example, theconductive oligomers depicted in Structure 13, 14 and 15 may be replacedby ETMs; alternatively, the ETMs may be added to the free terminus ofthe conductive oligomer.

In a preferred embodiment, a metallocene serves as the ETM, and isattached via an amide bond as depicted below in Structure 33. Theexamples outline the synthesis of a preferred compound when themetallocene is ferrocene.

In a preferred embodiment, amine linkages are used, as is generallydepicted in Structure 34.

Z is a linker, as defined herein, with 1-16 atoms being preferred, and2-4 atoms being particularly preferred, and t is either one or zero.

In a preferred embodiment, oxo linkages are used, as is generallydepicted in Structure 35.

In Structure 35, Z is a linker, as defined ETM herein, and t is eitherone or zero. Preferred Z linkers include alkyl groups includingheteroalkyl groups such as (CH₂)_(n) and (CH₂CH₂O)_(n), with n from 1 to10 being preferred, and n=1 to 4 being especially preferred, and n=4being particularly preferred.

Linkages utilizing other heteroatoms are also possible.

In a preferred embodiment, an ETM is attached to a phosphate at anyposition of the ribose-phosphate backbone of the nucleic acid. This maybe done in a variety of ways. In one embodiment, phosphodiester bondanalogs such as phosphoramide or phosphoramidite linkages may beincorporated into a nucleic acid, where the heteroatom (i.e. nitrogen)serves as a transition metal ligand (see PCT publication WO 95/15971,incorporated by reference). Alternatively, the conductive oligomersdepicted in Structures 23 and 24 may be replaced by ETMs. In a preferredembodiment, the composition has the structure shown in Structure 36.

In Structure 36, the ETM is attached via a phosphate linkage, generallythrough the use of a linker, Z. Preferred Z linkers include alkylgroups, including heteroalkyl groups such as (CH₂)_(n), (CH₂CH₂O)_(n),with n from 1 to 10 being preferred, and n=1 to 4 being especiallypreferred, and n=4 being particularly preferred.

When the ETM is attached to the base or the backbone of the nucleoside,it is possible to attach the ETMs via “dendrimer” structures, as is morefully outlined below. As is generally depicted in the Figures,alkyl-based linkers can be used to create multiple branching structurescomprising one or more ETMs at the terminus of each branch (althoughinternal ETMs can be used as well). Generally, this is done by creatingbranch points containing multiple hydroxy groups, which optionally canthen be used to add additional branch points. The terminal hydroxygroups can then be used in phosphoramidite reactions to add ETMs, as isgenerally done below for the nucleoside replacement and metallocenepolymer reactions. The branch point can be an internal one or a terminalone, and can be a chemical branch point or a nucleoside branch point.

In a preferred embodiment, an ETM such as a metallocene is used as a“nucleoside replacement”, serving as an ETM. For example, the distancebetween the two cyclopentadiene rings of ferrocene is similar to theorthongonal distance between two bases in a double stranded nucleicacid. Other metallocenes in addition to ferrocene may be used, forexample, air stable metallocenes such as those containing cobalt orruthenium. Thus, metallocene moieties may be incorporated into thebackbone of a nucleic acid, as is generally depicted in Structure 37(nucleic acid with a ribose-phosphate backbone) and Structure 38(peptide nucleic acid backbone). Structures 37 and 38 depict ferrocene,although as will be appreciated by those in the art, other metallocenesmay be used as well. In general, air stable metallocenes are preferred,including metallocenes utilizing ruthenium and cobalt as the metal.

In Structure 37, Z is a linker as defined above, with generally short,alkyl groups, including heteroatoms such as oxygen being preferred.Generally, what is important is the length of the linker, such thatminimal perturbations of a double stranded nucleic acid is effected, asis more fully described below. Thus, methylene, ethylene, ethyleneglycols, propylene and butylene are all preferred, with ethylene andethylene glycol being particularly preferred. In addition, each Z linkermay be the same or different. Structure 37 depicts a ribose-phosphatebackbone, although as will be appreciated by those in the art, nucleicacid analogs may also be used, including ribose analogs and phosphatebond analogs.

In Structure 38, preferred Z groups are as listed above, and again, eachZ linker can be the same or different. As above, other nucleic acidanalogs may be used as well.

In addition, although the structures and discussion above depictmetallocenes, and particularly ferrocene, this same general idea can beused to add ETMs in addition to metallocenes, as nucleoside replacementsor in polymer embodiments, described below. Thus, for example, when theETM is a transition metal complex other than a metallocene, comprisingone, two or three (or more) ligands, the ligands can be functionalizedas depicted for the ferrocene to allow the addition of phosphoramiditegroups. Particularly preferred in this embodiment are complexescomprising at least two ring (for example, aryl and substituted aryl)ligands, where each of the ligands comprises functional groups forattachment via phosphoramidite chemistry. As will be appreciated bythose in the art, this type of reaction, creating polymers of ETMseither as a portion of the backbone of the nucleic acid or as “sidegroups” of the nucleic acids, to allow amplification of the signalsgenerated herein, can be done with virtually any ETM that can befunctionalized to contain the correct chemical groups.

Thus, by inserting a metallocene such as ferrocene (or other ETMs) intothe backbone of a nucleic acid, nucleic acid analogs are made; that is,the invention provides nucleic acids having a backbone comprising atleast one metallocene. This is distinguished from nucleic acids havingmetallocenes attached to the backbone, i.e. via a ribose, a phosphate,etc. That is, two nucleic acids each made up of a traditional nucleicacid or analog (nucleic acids in this case including a singlenucleoside), may be covalently attached to each other via a metallocene.Viewed differently, a metallocene derivative or substituted metalloceneis provided, wherein each of the two aromatic rings of the metallocenehas a nucleic acid substitutent group.

In addition, as is more fully outlined below, it is possible toincorporate more than one metallocene into the backbone, either withnucleotides in between and/or with adjacent metallocenes. When adjacentmetallocenes are added to the backbone, this is similar to the processdescribed below as “metallocene polymers”; that is, there are areas ofmetallocene polymers within the backbone.

In addition to the nucleic acid substitutent groups, it is alsodesirable in some instances to add additional substituent groups to oneor both of the aromatic rings of the metallocene (or ETM). For example,as these nucleoside replacements are generally part of probe sequencesto be hybridized with a substantially complementary nucleic acid, forexample a target sequence or another probe sequence, it is possible toadd substitutent groups to the metallocene rings to facilitate hydrogenbonding to the base or bases on the opposite strand. These may be addedto any position on the metallocene rings. Suitable substitutent groupsinclude, but are not limited to, amide groups, amine groups, carboxylicacids, and alcohols, including substituted alcohols. In addition, thesesubstitutent groups can be attached via linkers as well, although ingeneral this is not preferred.

In addition, substituent groups on an ETM, particularly metallocenessuch as ferrocene, may be added to alter the redox properties of theETM. Thus, for example, in some embodiments, as is more fully describedbelow, it may be desirable to have different ETMs attached in differentways (i.e. base or ribose attachment), on different probes, or fordifferent purposes (for example, calibration or as an, internalstandard). Thus, the addition of substituent groups on the metallocenemay allow two different ETMs to be distinguished.

In order to generate these metallocene-backbone nucleic acid analogs,the intermediate components are also provided. Thus, in a preferredembodiment, the invention provides phosphoramidite metallocenes, asgenerally depicted in Structure 39:

In Structure 39, PG is a protecting group, generally suitable for use innucleic acid synthesis, with DMT, MMT and TMT all being preferred. Thearomatic rings can either be the rings of the metallocene, or aromaticrings of ligands for transition metal complexes or other organic ETMs.The aromatic rings may be the same or different, and may be substitutedas discussed herein. Structure 40 depicts the ferrocene derivative:

These phosphoramidite analogs can be added to standard oligonucleotidesyntheses as is known in the art.

Structure 41 depicts the ferrocene peptide nucleic acid (PNA) monomer,that can be added to PNA synthesis as is known in the art:

In Structure 41, the PG protecting group is OH suitable for use inpeptide nucleic acid synthesis, with MMT, boc and Fmoc being preferred.

These same intermediate compounds can be used to form ETM or metallocenepolymers, which are added to the nucleic acids, rather than as backbonereplacements, as is more fully described below.

In a preferred embodiment, the ETMs are attached as polymers, forexample as metallocene polymers, in a “branched” configuration similarto the “branched DNA” embodiments herein and as outlined in U.S. Pat.No. 5,124,246, using modified functionalized nucleotides. The generalidea is as follows. A modified phosphoramidite nucleotide is generatedthat can ultimately contain a free hydroxy group that can be used in theattachment of phosphoramidite ETMs such as metallocenes. This freehydroxy group could be on the base or the backbone, such as the riboseor the phosphate (although as will be appreciated by those in the art,nucleic acid analogs containing other structures can also be used). Themodified nucleotide is incorporated into a nucleic acid, and any hydroxyprotecting groups are removed, thus leaving the free hydroxyl. Upon theaddition of a phosphoramidite ETM such as a metallocene, as describedabove in structures 39 and 40, ETMs, such as metallocene ETMs, areadded. Additional phosphoramidite ETMs such as metallocenes can beadded, to form “ETM polymers”, including “metallocene polymers” asdepicted herein, particularly for ferrocene. In addition, in someembodiments, it is desirable to increase the solubility of the polymersby adding a “capping” group to the terminal ETM in the polymer, forexample a final phosphate group to the metallocene as is generallydepicted in FIG. 12. Other suitable solubility enhancing “capping”groups will be appreciated by those in the art. It should be noted thatthese solubility enhancing groups can be added to the polymers in otherplaces, including to the ligand rings, for example on the metallocenesas discussed herein

In a preferred embodiment, (as depicted in the figures of U.S. Ser. No.09/626,096) the 2′ position of a ribose of a phosphoramidite nucleotideis first functionalized to contain a protected hydroxy group, in thiscase via an oxo-linkage, although any number of linkers can be used, asis generally described herein for Z linkers. The protected modifiednucleotide is then incorporated via standard phosphoramidite chemistryinto a growing nucleic acid. The protecting group is removed, and thefree hydroxy group is used, again using standard phosphoramiditechemistry to add a phosphoramidite metallocene such as ferrocene. Asimilar reaction is possible for nucleic acid analogs. For example,using peptide nucleic acids and the metallocene monomer shown inStructure 41, peptide nucleic acid structures containing metallocenepolymers could be generated.

Thus, the present invention provides recruitment linkers of nucleicacids comprising “branches” of metallocene polymers as is generallydepicted in FIGS. 12 and 13. Preferred embodiments also utilizemetallocene polymers from one to about 50 metallocenes in length, withfrom about 5 to about 20 being preferred and from about 5 to about 10being especially preferred.

In addition, when the recruitment linker is nucleic acid, anycombination of ETM attachments may be done.

In a preferred embodiment, the recruitment linker is not nucleic acid,and instead may be any sort of linker or polymer. As will be appreciatedby those in the art, generally any linker or polymer that can bemodified to contain ETMs can be used. In general, the polymers orlinkers should be reasonably soluble and contain suitable functionalgroups for the addition of ETMs.

As used herein, a “recruitment polymer” comprises at least two or threesubunits, which are covalently attached. At least some portion of themonomeric subunits contain functional groups for the covalent attachmentof ETMs. In some embodiments coupling moieties are used to covalentlylink the subunits with the ETMs. Preferred functional groups forattachment are amino groups, carboxy groups, oxo groups and thiolgroups, with amino groups being particularly preferred. As will beappreciated by those in the art, a wide variety of recruitment polymersare possible.

Suitable linkers include, but are not limited to, alkyl linkers(including heteroalkyl (including (poly)ethylene glycol-typestructures), substituted alkyl, aryalkyl linkers, etc. As above for thepolymers, the linkers will comprise one or more functional groups forthe attachment of ETMs, which will be done as will be appreciated bythose in the art, for example through the use homo- orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference).

Suitable recruitment polymers include, but are not limited to,functionalized styrenes, such as amino styrene, functionalized dextrans,and polyamino acids. Preferred polymers are polyamino acids (bothpoly-D-amino acids and poly-L-amino acids), such as polylysine, andpolymers containing lysine and other amino acids being particularlypreferred. Other suitable polyamino acids are polyglutamic acid,polyaspartic acid, co-polymers of lysine and glutamic or aspartic acid,co-polymers of lysine with alanine, tyrosine, phenylalanine, serine,tryptophan, and/or proline.

In a preferred embodiment, the recruitment linker comprises ametallocene polymer, as is described above.

The attachment of the recruitment linkers to the first portion of thelabel probe will depend on the composition of the recruitment linker, aswill be appreciated by those in the art. When the recruitment linker isnucleic acid, it is generally formed during the synthesis of the firstportion of the label probe, with incorporation of nucleosides containingETMs as required. Alternatively, the first portion of the label probeand the recruitment linker may be made separately, and then attached.For example, there may be an overlapping section of complementarity,forming a section of double stranded nucleic acid that can then bechemically crosslinked, for example by using psoralen as is known in theart.

When non-nucleic acid recruitment linkers are used, attachment of thelinker/polymer of the recruitment linker will be done generally usingstandard chemical techniques, such as will be appreciated by those inthe art. For example, when alkyl-based linkers are used, attachment canbe similar to the attachment of insulators to nucleic acids.

In addition, it is possible to have recruitment linkers that aremixtures of nucleic acids and non-nucleic acids, either in a linear form(e.g. nucleic acid segments linked together with alkyl linkers) or inbranched forms (nucleic acids with alkyl “branches” that may containETMs and may be additionally branched).

In a preferred embodiment, it is the target sequence itself that carriesthe ETMs, rather than the recruitment linker of a label probe. Forexample, as is more fully described below, it is possible toenzymatically add triphosphate nucleotides comprising the ETMs of theinvention to a growing nucleic acid, for example during a polymerasechain reaction (PCR). As will be recognized by those in the art, whileseveral enzymes have been shown to generally tolerate modifiednucleotides, some of the modified nucleotides of the invention, forexample the “nucleoside replacement” embodiments and putatively some ofthe phosphate attachments, may or may not be recognized by the enzymesto allow incorporation into a growing nucleic acid. Therefore, preferredattachments in this embodiment are to the base or ribose of thenucleotide.

Thus, for example, PCR amplification of a target sequence, as is wellknown in the art, will result in target sequences comprising ETMs,generally randomly incorporated into the sequence. The system of theinvention can then be configured to allow detection using these ETMs, asis generally depicted in FIGS. 16A, 16B and 16D of U.S. Ser. No.60/190,259.

Alternatively, as outlined more fully below, it is possible toenzymatically add nucleotides comprising ETMs to the terminus of anucleic acid, for example a target nucleic acid. In this embodiment, aneffective “recruitment linker” is added to the terminus of the targetsequence, that can then be used for detection. Thus the inventionprovides compositions utilizing electrodes comprising monolayers ofconductive oligomers and capture probes, and target sequences thatcomprise a first portion that is capable of hybridizing to a componentof an assay complex, and a second portion that does not hybridize to acomponent of an assay complex and comprises at least one covalentlyattached electron transfer moiety. Similarly, methods utilizing thesecompositions are also provided.

It is also possible to have ETMs connected to probe sequences, i.e.sequences designed to hybridize to complementary sequences. Thus, ETMsmay be added to non-recruitment linkers as well. For example, there maybe ETMs added to sections of label probes that do hybridize tocomponents of the assay complex, for example the first portion, or tothe target sequence as outlined above. These ETMs may be used forelectron transfer detection in some embodiments, or they may not,depending on, the location and system. For example, in some embodiments,when for example the target sequence containing randomly incorporatedETMs is hybridized directly to the capture probe, as is depicted in FIG.16A of U.S. Ser. No. 60/190,259, there may be ETMs in the portionhybridizing to the capture probe. If the capture probe is attached tothe electrode using a conductive oligomer, these ETMs can be used todetect electron transfer as has been previously described.Alternatively, these ETMs may not be specifically detected.

Similarly, in some embodiments, when the recruitment linker is nucleicacid, it may be desirable in some instances to have some or all of therecruitment linker be double stranded. In one embodiment, there may be asecond recruitment linker, substantially complementary to the firstrecruitment linker, that can hybridize to the first recruitment linker.In a preferred embodiment, the first recruitment linker comprises thecovalently attached ETMs. In an alternative embodiment, the secondrecruitment linker contains the ETMs, and the first recruitment linkerdoes not, and the ETMs are recruited to the surface by hybridization ofthe second recruitment linker to the first. In yet another embodiment,both the first and second recruitment linkers comprise ETMs. It shouldbe noted, as discussed above, that nucleic acids comprising a largenumber of ETMs may not hybridize as well, i.e. the T_(m) may bedecreased, depending on the site of attachment and the characteristicsof the ETM. Thus, in general, when multiple ETMs are used on hybridizingstrands, generally there are less than about 5, with less than about 3being preferred, or alternatively the ETMs should be spaced sufficientlyfar apart that the intervening nucleotides can sufficiently hybridize toallow good kinetics.

In one embodiment, non-covalently attached ETMs may be used. In oneembodiment, the ETM is a hybridization indicator. Hybridizationindicators serve as ETMs that will preferentially associate with doublestranded nucleic acid, usually reversibly, similar to the method ofMillan et al., Anal. Chem. 65:2317-2323 (1993); Millan et al., Anal.Chem. 662943-2948 (1994), both of which are hereby expresslyincorporated by reference. In this embodiment, increases in the localconcentration of ETMs, due to the association of the ETM hybridizationindicator with double stranded nucleic acid at the surface, can bemonitored using the monolayers comprising the conductive oligomers.Hybridization indicators include intercalators and minor and/or majorgroove binding moieties. In a preferred embodiment, intercalators may beused; since intercalation generally only occurs in the presence ofdouble stranded nucleic acid, only in the presence of double strandednucleic acid will the ETMs concentrate. Intercalating transition metalcomplex ETMs are known in the art. Similarly, major or minor groovebinding moieties, such as methylene blue, may also be used in thisembodiment.

Similarly, the systems of the invention may utilize non-covalentlyattached ETMs, as is generally described in Napier et al., Bioconj.Chem. 8:906 (1997), hereby expressly incorporated by reference. In thisembodiment, changes in the redox state of certain molecules as a resultof the presence of DNA (i.e. guanine oxidation by ruthenium complexes)can be detected using SAMs comprising conductive oligomers.

Thus, the present invention provides electrodes comprising monolayerscomprising conductive oligomers, generally including capture probes, andeither target sequences or label probes comprising recruitment linkerscontaining ETMs. Probes of the present invention are designed to becomplementary to a target sequence (either the target sequence of thesample or to other probe sequences, as is described below), such thathybridization of the target sequence and the probes of the presentinvention occurs. As outlined below, this complementarity need not beperfect; there may be any number of base pair mismatches which willinterfere with hybridization between the target sequence and the singlestranded nucleic acids of the present invention. However, if the numberof mutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, by “substantially complementary”herein is meant that the probes are sufficiently complementary to thetarget sequences to hybridize under normal reaction conditions.

Generally, the nucleic acid compositions of the invention are useful asoligonucleotide probes. As is appreciated by those in the art, thelength of the probe will vary with the length of the target sequence andthe hybridization and wash conditions. Generally, oligonucleotide probesrange from about 8 to about 50 nucleotides, with from about 10 to about30 being preferred and from about 12 to about 25 being especiallypreferred. In some cases, very long probes may be used, e.g. 50 to200-300 nucleotides in length. Thus, in the structures depicted herein,nucleosides may be replaced with nucleic acids.

A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions; seefor example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2dEdition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, etal, hereby incorporated by reference. The hybridization conditions mayalso vary when a non-ionic backbone, e.g. PNA is used, as is known inthe art. In addition, cross-linking agents may be added after targetbinding to cross-link, i.e. covalently attach, the two strands of thehybridization complex.

As will be appreciated by those in the art, the systems of the inventionmay take on a large number of different configurations, as is generallydepicted in the Figures of U.S. Ser. No. 09/626,096 (the Figures in thenext paragraphs refer to the figures of U.S. Ser. No. 09/626,096). Ingeneral, there are three types of systems that can be used: (1) systemsin which the target sequence itself is labeled with ETMs (see FIGS. 16A,16B and 16D); (2) systems in which label probes directly hybridize tothe target sequences (see FIGS. 16C and 16H); and (3) systems in whichlabel probes are indirectly hybridized to the target sequences, forexample through the use of amplifier probes (see FIGS. 16E, 16F and16G).

In general, for all the systems outlined herein, both for nucleic acidsand other target analytes, the invention provides assay complexes thatminimally comprise a target analyte and a capture binding ligand. Fornucleic acid target sequences, by “assay complex” herein is meant thecollection of hybridization complexes comprising nucleic acids,including probes and targets, that contains at least one label(preferably an ETM in the electronic methods of the present invention)and thus allows detection. The composition of the assay complex dependson the use of the different probe components outlined herein. The assaycomplexes may also include label probes, capture extender probes, labelextender probes, and amplifier probes, as outlined herein and in U.S.Ser. No. 09/626,096, depending on the configuration used.

The assays are generally run under stringency conditions which allowformation of the label probe hybridization complex only in the presenceof target. Stringency can be controlled by altering a step parameterthat is a thermodynamic variable, including, but not limited to,temperature, formamide concentration, salt concentration, chaotropicsalt concentration pH, organic solvent concentration, etc.

These parameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirableto perform certain steps at higher stringency conditions; for example,when an initial hybridization step is done between the target sequenceand the label extender and capture extender probes. Running this step atconditions which favor specific binding can allow the reduction ofnon-specific binding.

The reactions outlined herein may be accomplished in a variety of ways,as will be appreciated by those in the art. Components of the reactionmay be added simultaneously, or sequentially, in any order, withpreferred embodiments outlined below. In addition, the reaction mayinclude a variety of other reagents. These include reagents like salts,buffers, neutral proteins (e.g. albumin), detergents, etc which may beused to facilitate optimal hybridization and detection, and/or reducenon-specific or background interactions. Also reagents that otherwiseimprove the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc., may be used, dependingon the sample preparation methods and purity of the target.

Accordingly, the present invention provides biochips, with covalentlyattached capture binding ligands (e.g. capture probes). The biochips areincorporated into the cartridges of the invention and then fitted intothe stations of the multiplexing devices of the invention for runningassays.

In a preferred embodiment, the biochips are attached to the rest of thecartridge in a wide variety of ways. In one embodiment, the biochip ismade directly on a portion of the cartridge and is thus incorporatedinto the system. Alternatively, as outlined herein, when the biochip isformulated on a different substrate than the remainder of the cartridge,there are a variety of attachment mechanisms that can be used, dependingon the composition and configuration of the two substrates. For example,when the biochip is formulated on printed circuit board material, therecan be “pins” or “rods” that are inserted into holes, with subsequentfusion (for example, using solvents or heat). Similarly,surface-to-surface heat or solvent fusion may be done. Alternatively,adhesives can be used to glue the two together. Similarly, thesetechniques can be used with additional sealing components such asgaskets. Alternatively, the biochip may “snap” into the cartridge, usingcomponents such as molded plastic snapping devices.

The present invention further provides for holders for the cartridgesfor loading with samples, prior to loading the cartridges into thestations of the device. In general, as will be appreciated by those inthe art, the holders may be configured in a wide variety of ways,depending on the configuration of the cartridges and caps, if present.For example, holders that align cartridges such that standard reagenthandling tools can be used are preferred. As shown in the Figures,holders that allow the use of multichannel pipettemen or robotic systemsbased on 96 well formats are preferred. The holders may also include thecaps, positioned for easy use, or reagents and/or buffer components. Ingeneral, the holders are fabricated out of materials resistant to thechemicals and reagents used in the assays.

The cartridges of the invention are designed to be inserted intostations in a multiplexing device. As will be appreciated by those inthe art and described below, the devices of the invention can take on awide variety of conformations, depending on the desired components, theend use, the ultimate desired size of the instrument, etc.

Each multiplexing device has a number of different stations into whichthe cartridges are inserted. The cartridge/station pair can beconfigured in a variety of ways to include the use of “snap-in” locks,asymmetry such that the cartridge only fits into the device in aparticular orientation, different size stations for different sizecartridges (for example, some rare amount of tests may require specialhandling and the machines may be designed with special stations forthese tests). This embodiment may also utilize electronic sensors thatdetect the presence or absence of a cartridge, or whether the cartridgeis correctly positioned.

In general, the number of stations per device will vary with the desireduse. Preferred embodiments utilize at least two or three stations, withat least 5-100 being preferred, and from about 25-50 being particularlypreferred, with 48 being especially preferred. In general, the devicesare laid out as a matrix, with columns and rows of stations.

As outlined herein, each station can have a number of differentfunctional components, including, but not limited to, interconnects toelectronic components, thermocontrollers, signaling systems, sensors forleak detection, alphanumeric displays, and detectors.

In a preferred embodiment, when the cartridge comprises a biochip thatrelies on electrodes for detection, the stations comprise matchinginterconnects for the biochip, to allow electronic communication betweenthe chip and the device.

In a preferred embodiment, each station comprises an individual thermalcontroller. “Thermal controller” or “thermocontroller” in this contextincludes elements that can both heat and cool the cartridges and thusthe samples in the cartridges as well. In general, given the size andfunction of the systems, it is desirable to utilize small, fastthermocontrollers. There are a wide variety of known suitablethermocontrollers, including Peltier systems.

In general, the thermocontroller should be able to heat cool samplesranging from 0 to about 100° C. and at a rate ranging from 0.01° C./secto 10° C./sec.

It should be noted that a thermocontroller can be used after an assay todestroy the biological material in the cartridge. That is, it isfrequently desirable to minimize the exposure of health care workers andlab workers to potentially dangerous samples, and to facilitate thedisposal of these materials. The thermocontroller can be used to heatthe spent sample at extreme temperatures for some period of time inorder to kill or destroy the sample. In addition, heating in conjunctionwith the addition of other generally harsh reagents (strong acid, strongbase, etc.) can also be used. Furthermore, in some embodiments, an RFantennae is used to generate plasma that is pumped into the chamberafter fluid evacuation to destroy all biological material.

In one embodiment, rather than each station comprising an individualthermal controller, sets (for example, rows or columns) of the stationsshare a thermal controller. In an alternative embodiment, themultiplexing device comprises a single thermal controller.

In a preferred embodiment, the devices of the invention include a “StatSlot”, where a cartridge can be put in and read right away at onestation, rather than run as a sequence. In general, the temperature atthis station may be preset.

In a preferred embodiment, the stations of the device include signalingsystems. For example, a system of lights, particularly colored lights,at each station can be used to indicate the status of the cartridge orthe assay: cartridge present or absent, assay in progress, error, assaycompleted, etc. In addition, the configuration of the lights may be thecode (particularly for color blind people); two lights for cartridge in,flashing lights for assay finished, etc. Again, these signaling systemsmay be at each station or at sets of stations.

In a preferred embodiment, the devices of the invention include analphanumeric display to allow the display of data or other information.For example, this display may be used in conjunction with a barcodereader, described below, to show the operator which cartridge wasinserted (e.g. the HIV panel, the HCV panel, the infectious diseasepanel, the breast cancer SNP panel, etc.), or other data about thecartridge (lot or batch number, etc.). In addition, the display can beused to give the operator the test results, etc. As for the signalingsystems, a display can be at each station, or there may be displays forsets of stations or for the whole device.

In a preferred embodiment, each station of the device may be configuredto allow electrophoresis or dielectrophoresis on the biochip. That is,as is generally described in WO99/67425 and U.S. Ser. No. 09/171,981,hereby incorporated by reference, there may be additional electrodes orelectronic components to allow the concentration and/or movement ofanalytes to the surface of the array. Similarly, as is described inWO99/67425 and U.S. Ser. No. 09/171,981, the electrophoresis ordielectrophoresis electrodes may be contained on the biochip.

In a preferred embodiment, the device (or alternatively, each station)comprises a barcode reader to read a corresponding barcode on thecartridge. These barcodes may be used for a wide variety of purposes,including, but not limited to, identifying the sample (e.g. patientnumber or code), the test being done, the batch number of the chip,calibration information, assay protocols including cycle time, signalprocessing requirements, etc.

In addition, the barcode can be used to control the instrument. Forexample, instrument control may be through the use of a keyboard, amouse or a barcode reader. Thus, for example, there may be barcodes onthe cartridges to indicate the identity of the chip, but also on a cardto scan for starting the assay, stopping the assay, downloading thedata, etc. In a preferred embodiment, the card of barcode commands arefound in a drawer or storage compartment of the device, outlined herein.

In a preferred embodiment, each station comprises a memory chip reader.Again, in this embodiment, each cartridge comprises a memory chip, thatcan have sample information (e.g. patient number or code), the testbeing done, the batch number of the chip, calibration information, assayprotocols, etc.), or what the user interface looks like (for example,not a number but “HIV positive”), etc.

In a preferred embodiment, each station comprises a memory chip writerto add information to the cartridge, such as what test was done, thedate, the results, etc.

In a preferred embodiment, each station has encryption components inconjunction with the cartridge, to encrypt patient information. There isa growing concern regarding the confidentiality of patient information,particularly with regard to employment and insurance issues. Thus forexample, in some embodiments, the devices of the invention will notallow the operator to know the results of the test. Rather, the outputwill be a confirmation that the test was performed correctly and aviable answer received, but will say nothing about the actual test beingdone or the results. The test results themselves, in addition to thepatient information, can be encrypted and sent to a remote location asoutlined below for processing, decryption or storage.

In a preferred embodiment, the device may include drawers or storagecompartments to allow the storage of reagents, cartridges, caps,holders, pipettemen, etc.

In a preferred embodiment, for example, when fluorescence dyes are usedin the assays, fluorescent readers are used. In one embodiment, thedevice comprises a reader at each station. Alternatively, in a preferredembodiment, the device comprises a single reader that is moved, eitherby moving the reader or by moving the stations to a single reader withinthe device. Thus, in some embodiments, there are motors, pulleys, cords,etc. to allow the movement of stations, cartridges or readers.

In a preferred embodiment, the devices of the invention comprise liquidhandling components, including components for loading and unloadingfluids at each station or sets of stations. The liquid handling systemscan include robotic systems comprising any number of components. Inaddition, any or all of the steps outlined herein may be automated;thus, for example, the systems may be completely or partially automated.

As will be appreciated by those in the art, there are a wide variety ofcomponents which can be used, including, but not limited to, one or morerobotic arms; plate handlers for the positioning of microplates; holderswith cartridges and/or caps; automated lid or cap handlers to remove andreplace lids for wells on non-cross contamination plates; tip assembliesfor sample distribution with disposable tips; washable tip assembliesfor sample distribution; 96 well loading blocks; cooled reagent racks;microtiter plate pipette positions (optionally cooled); stacking towersfor plates and tips; and computer systems.

Fully robotic or microfluidic systems include automated liquid-,particle-, cell- and organism-handling including high throughputpipetting to perform all steps of screening applications. This includesliquid, particle, cell, and organism manipulations such as aspiration,dispensing, mixing, diluting, washing, accurate volumetric transfers;retrieving, and discarding of pipet tips; and repetitive pipetting ofidentical volumes for multiple deliveries from a single sampleaspiration. These manipulations are cross-contamination-free liquid,particle, cell, and organism transfers. This instrument performsautomated replication of microplate samples to filters, membranes,and/or daughter plates, high-density transfers, full-plate serialdilutions, and high capacity operation.

In a preferred embodiment, chemically derivatized particles, plates,cartridges, tubes, magnetic particles, or other solid phase matrix withspecificity to the assay components are used. The binding surfaces ofmicroplates, tubes or any solid phase matrices include non-polarsurfaces, highly polar surfaces, modified dextran coating to promotecovalent binding, antibody coating, affinity media to bind fusionproteins or peptides, surface-fixed proteins such as recombinant proteinA or G, nucleotide resins or coatings, and other affinity matrix areuseful in this invention.

In a preferred embodiment, platforms for multi-well plates, multi-tubes,holders, cartridges, minitubes, deep-well plates, microfuge tubes,cryovials, square well plates, filters, chips, optic fibers, beads, andother solid-phase matrices or platform with various volumes areaccommodated on an upgradable modular platform for additional capacity.This modular platform includes a variable speed orbital shaker, andmulti-position work decks for source samples, sample and reagentdilution, assay plates, sample and reagent reservoirs, pipette tips, andan active wash station.

In a preferred embodiment, thermocycler and thermoregulating systems areused for stabilizing the temperature of heat exchangers such ascontrolled blocks or platforms to provide accurate temperature controlof incubating samples from 0 BC to 100 BC; this is in addition to or inplace of the station thermocontrollers.

In a preferred embodiment, interchangeable pipet heads (single ormulti-channel) with single or multiple magnetic probes, affinity probes,or pipetters robotically manipulate the liquid, particles, cells, andorganisms. Multi-well or multi-tube magnetic separators or platformsmanipulate liquid, particles, cells, and organisms in single or multiplesample formats.

In some embodiments, for example when electronic detection is not done,the instrumentation will include a detector, which can be a wide varietyof different detectors, depending on the labels and assay. In apreferred embodiment, useful detectors include a microscope(s) withmultiple channels of fluorescence; plate readers to provide fluorescent,ultraviolet and visible spectrophotometric detection with single anddual wavelength endpoint and kinetics capability, fluoroescenceresonance energy transfer (FRET), luminescence, quenching, two-photonexcitation, and intensity redistribution; CCD cameras to capture andtransform data and images into quantifiable formats; and a computerworkstation.

These instruments can fit in a sterile laminar flow or fume hood, or areenclosed, self-contained systems, for cell culture growth andtransformation in multi-well plates or tubes and for hazardousoperations. The living cells may be grown under controlled growthconditions, with controls for temperature, humidity, and gas for timeseries of the live cell assays. Automated transformation of cells andautomated colony pickers may facilitate rapid screening of desiredcells.

Flow cytometry or capillary electrophoresis formats can be used forindividual capture of magnetic and other beads, particles, cells, andorganisms.

The flexible hardware and software allow instrument adaptability formultiple applications. The software program modules allow creation,modification, and running of methods. The system diagnostic modulesallow instrument alignment, correct connections, and motor operations.The customized tools, labware, and liquid, particle, cell and organismtransfer patterns allow different applications to be performed. Thedatabase allows method and parameter storage. Robotic and computerinterfaces allow communication between instruments.

In a preferred embodiment, the robotic apparatus includes a centralprocessing unit which communicates with a memory and a set ofinput/output devices (e.g., keyboard, mouse, monitor, printer, etc.)through a bus. Again, as outlined below, this may be in addition to orin place of the CPU for the multiplexing devices of the invention. Thegeneral interaction between a central processing unit, a memory,input/output devices, and a bus is known in the art. Thus, a variety ofdifferent procedures, depending on the experiments to be run, are storedin the CPU memory.

These robotic fluid handling systems can utilize any number of differentreagents, including buffers, reagents, samples, washes, assay componentssuch as label probes, etc.

In a preferred embodiment, the devices of the invention include sensorsfor leak detection. These are generally of two types; either electronicmeasurements of resistance or the spiking of the assay with optical ordetectable tags. This may be particularly important in some embodimentswhere biohazardous materials or caustic chemicals are being tested.

In a preferred embodiment, the devices of the invention comprise adevice board that can be used to do a variety of analyses, includingsignal processing, digital lock-in, comprising logic circuits, etc., asoutlined herein.

In a preferred embodiment, the devices of the invention comprise adevice board that can be used to do a variety of analyses, includingsignal processing, digital lock-in, comprising logic circuits, etc., asoutlined herein.

In a preferred embodiment, the systems of the invention comprise aprocessor or central processing unit (CPU) with an associated memory.The associated memory can be memory on-board the processor and optionalmemory coupled to the processor via an external memory bus. Thisprocessor (CPU) can be physically contained within the apparatus itself,can be connected to the apparatus via a cable, or can be connected usingwireless technology. There can be one or more per device or one can beshared among devices.

For example, in one embodiment the systems of the invention provide amotherboard on which are mounted the CPU and associated memory. Themotherboard may desirably provide connectors for mechanically andelectrically connecting with one or a plurality of edge connectormountable printed circuit cards having the signal processing circuitsformed thereon. The edge connectors provide signal and power connectionswith the motherboard. In one embodiment, the CPU and memory, inconjunction with an operating system, support the menu or command drivenoperation and analysis described elsewhere herein. Software and/orfirmware executing in the processor and/or within the signal processingprinted circuit card components are used to control the operation of theapparatus, devices, and system.

In yet another embodiment, the system of the invention are configuredand operate in the manner of a computer peripheral device coupled to anexternal personal computer. In this type of implementation, each signalprocessing printed circuit card may be connected to the personalcomputer by a separate communication channel or link (such as forexample, by one or more serial, parallel, SCSI, Fire-wire, blue-tooth,or other wired or wireless communication channel or link), or multiplesignal processing printed circuit cards may be multiplexed to share asmaller number of communication channels or links. Typically, themultiple signal processing printed circuit cards will interconnect via acommunication bus, such as may be provided by a motherboard or otherinterconnect structure. Each signal processing board may have a uniqueaddress (locally or globally unique) such that communications directedbetween signal processing cards or between a signal processing printedcircuit card to the processor may be identified with the signalprocessing card and interpreted and/or routed accordingly. The PCcontains an application program that controls the instrument andcollects data from the instrument. Those workers having ordinary skillin the art in light of the description provided here will appreciatethat there are numerous ways of connecting specialized instrumentationusing digital and/or analog circuits, such as the devices and apparatusdescribe here, and therefore not described in greater detail here.

In one particular embodiment, an apparatus having six sensor slots oneach of eight separate printed circuit based signal processing cards iscoupled with a personal computer via at least one serial interface, suchas an RS-232 or RS-485 serial link. Advantageously, a plurality of suchserial ports on each of the personal computer and apparatus are providedto increase bandwidth. In one embodiment, three serial input/outputinterfaces are provided.

In still another embodiment, a processor or CPU with associated memorymay be provided directly one each signal processing printed board.

In a preferred embodiment, the devices of the invention include alocalization device, such as a Global Positioning System (GPS) as areknown in the art. This may find particular use in agriculture andbiowarfare uses, as well as remote diagnosis of problems.

In a preferred embodiment, the devices of the invention includecomponents for the communication of data, assay results, patientinformation, etc. to an off-device location. Thus, for example, one ormore modems (including both telephone and cable modems), internet cards,infrared ports, etc. may be included in the devices to allow thetransmission of data and other relevant information (barcodeinformation, assay conditions and protocols, operator identification,time stamps, etc.) to a remote location such as a general informationrepository, hospitals, doctor's offices, epidemiology centers,pharmacies, government centers, insurance providers, etc.

In a preferred embodiment, the devices of the invention includecomponents for wireless communication systems, to allow thistransmission of data in the absence of physical electronic orcommunications connections. In addition, wireless receivers can beincluded.

Accordingly, the present invention provides methods and compositions forthe multiplex analysis of samples and target analytes. Samples (eitherraw samples or treated samples (e.g. amplified, purified, etc.)) areloaded into the cartridges of the invention, optional caps are put on,and the cartridges loaded into a station of the device. Additionalreagents are added as necessary, and assay complexes formed.

Once the assay complexes of the invention are made, that minimallycomprise a target sequence and a label probe, detection proceeds withelectronic initiation. Without being limited by the mechanism or theory,detection is based on the transfer of electrons from the ETM to theelectrode.

Detection of electron transfer, i.e. the presence of the ETMs, isgenerally initiated electronically, with voltage being preferred. Apotential is applied to the assay complex. Precise control andvariations in the applied potential can be via a potentiostat and eithera three electrode system (one reference, one sample (or working) and onecounter electrode) or a two electrode system (one sample and one counterelectrode). This allows matching of applied potential to peak potentialof the system which depends in part on the choice of ETMs and in part onthe other system components, the composition and integrity of themonolayer, and what type of reference electrode is used. As describedherein, ferrocene is a preferred ETM.

In some embodiments, co-reductants or co-oxidants are used as isgenerally described in WO00/16089, hereby expressly incorporated byreference.

The presence of the ETMs at the surface of the monolayer can be detectedin a variety of ways. A variety of detection methods may be used,including, but not limited to, optical detection (as a result ofspectral changes upon changes in redox states), which includesfluorescence, phosphorescence, luminiscence, chemiluminescence,electrochemiluminescence, and refractive index; and electronicdetection, including, but not limited to, amperommetry, voltammetry,capacitance and impedence. These methods include time or frequencydependent methods based on AC or DC currents, pulsed methods, lock-intechniques, filtering (high pass, low pass, band pass), andtime-resolved techniques including time-resolved fluorescence.

In one embodiment, the efficient transfer of electrons from the ETM tothe electrode results in stereotyped changes in the redox state of theETM. With many ETMs including the complexes of ruthenium containingbipyridine, pyridine and imidazole rings, these changes in redox stateare associated with changes in spectral properties. Significantdifferences in absorbance are observed between reduced and oxidizedstates for these molecules. See for example Fabbrizzi et al., Chem. Soc.Rev. 1995 pp 197-202). These differences can be monitored using aspectrophotometer or simple photomultiplier tube device.

In this embodiment, possible electron donors and acceptors include allthe derivatives listed above for photoactivation or initiation.Preferred electron donors and acceptors have characteristically largespectral changes upon oxidation and reduction resulting in highlysensitive monitoring of electron transfer. Such examples includeRu(NH₃)₄py and Ru(bpy)₂im as preferred examples. It should be understoodthat only the donor or acceptor that is being monitored by absorbanceneed have ideal spectral characteristics.

In a preferred embodiment, the electron transfer is detectedfluorometrically. Numerous transition metal complexes, including thoseof ruthenium, have distinct fluorescence properties. Therefore, thechange in redox state of the electron donors and electron acceptorsattached to the nucleic acid can be monitored very sensitively usingfluorescence, for example with Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺. Theproduction of this compound can be easily measured using standardfluorescence assay techniques. For example, laser induced fluorescencecan be recorded in a standard single cell fluorimeter, a flow through“on-line” fluorimeter (such as those attached to a chromatographysystem) or a multi-sample “plate-reader” similar to those marketed for96-well immuno assays.

Alternatively, fluorescence can be measured using fiber optic-sensorswith nucleic acid probes in solution or attached to the fiber optic.Fluorescence is monitored using a photomultiplier tube or other lightdetection instrument attached to the fiber optic. The advantage of thissystem is the extremely small volumes of sample that can be assayed.

In addition, scanning fluorescence detectors such as the FluorImagersold by Molecular Dynamics are ideally suited to monitoring thefluorescence of modified nucleic acid molecules arrayed on solidsurfaces. The advantage of this system is the large number of electrontransfer probes that can be scanned at once using chips covered withthousands of distinct nucleic acid probes.

Many transition metal complexes display fluorescence with large Stokesshifts. Suitable examples include bis- and trisphenanthroline complexesand bis- and trisbipyridyl complexes of transition metals such asruthenium (see Juris, A., Balzani, V., et. al. Coord. Chem. Rev., V. 84,p. 85-277, 1988). Preferred examples display efficient fluorescence(reasonably high quantum yields) as well as low reorganization energies.These include Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺,Ru(4,4′-diphenyl-2,2′-bipyridine)₃ ²⁺ and platinum complexes (seeCummings et al., J. Am. Chem. Soc. 118:1949-1960 (1996), incorporated byreference). Alternatively, a reduction in fluorescence associated withhybridization can be measured using these systems.

In a further embodiment, electrochemiluminescence is used as the basisof the electron transfer detection. With some ETMs such as Ru²⁺(bpy)₃,direct luminescence accompanies excited state decay. Changes in thisproperty are associated with nucleic acid hybridization and can bemonitored with a simple photomultiplier tube arrangement (see Blackburn,G. F. Clin. Chem. 37: 1534-1539 (1991); and Juris et al., supra.

In a preferred embodiment, electronic detection is used, includingamperommetry, voltammetry, capacitance, and impedence. Suitabletechniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltammetry (cyclic voltammetry, pulse voltammetry(normal pulse voltammetry, square wave voltammetry, differential pulsevoltammetry, Osteryoung square wave voltammetry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; AC voltammetry; and photoelectrochemistry.

In a preferred embodiment, monitoring electron transfer is viaamperometric detection. This method of detection involves applying apotential (as compared to a separate reference electrode) between thenucleic acid-conjugated electrode and a reference (counter) electrode inthe sample containing target genes of interest. Electron transfer ofdiffering efficiencies is induced in samples in the presence or absenceof target nucleic acid; that is, the presence or absence of the targetnucleic acid, and thus the label probe, can result in differentcurrents.

The device for measuring electron transfer amperometrically involvessensitive current detection and includes a means of controlling thevoltage potential, usually a potentiostat. This voltage is optimizedwith reference to the potential of the electron donating complex on thelabel probe. Possible electron donating complexes include thosepreviously mentioned with complexes of iron, osmium, platinum, cobalt,rhenium and ruthenium being preferred and complexes of iron being mostpreferred.

In a preferred embodiment, alternative electron detection modes areutilized. For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors are used tomonitor electron transfer between the ETM and the electrode. Inaddition, other properties of insulators and of conductors (such asresistance conductivity, impedance and capicitance) could be used tomonitor electron transfer between ETM and the electrode. Finally, anysystem that generates a current (such as electron transfer) alsogenerates a small magnetic field, which may be monitored in someembodiments.

It should be understood that one benefit of the fast rates of electrontransfer observed in the compositions of the invention is that timeresolution can greatly enhance the signal-to-noise results of monitorsbased on absorbance, fluorescence and electronic current. The fast ratesof electron transfer of the present invention result both in highsignals and stereotyped delays between electron transfer initiation andcompletion. By amplifying signals of particular delays, such as throughthe use of pulsed initiation of electron transfer and “lock-in”amplifiers of detection, and Fourier transforms.

In a preferred embodiment, electron transfer is initiated usingalternating current (AC) methods. Without being bound by theory, itappears that ETMs, bound to an electrode, generally respond similarly toan AC voltage across a circuit containing resistors and capacitors.

There are a variety of techniques that can be used to increase thesignal, decrease the noise, or make the signal more obvious ordetectable in a background of noise. That is, any technique that canserve to better identify a signal in the background noise may find usein the present invention. These techniques are generally classified inthree ways: (1) variations in the type or methods of applying theinitiation signals (i.e. varying the “input” to maximize or identify thesample signal); (2) data processing, i.e. techniques used on the“output” signals to maximize or identify the sample signal; and (3)variations in the assay itself, i.e. to the electrode surface or to thecomponents of the system, that allow for better identification of thesample signal. Thus, for example, suitable “input” AC methods include,but are not limited to, using multiple frequencies; increasing the ACamplitude; the use of square wave ACV; the use of special or complicatedwaveforms; etc. Similarly, suitable “output” AC techniques include, butare not limited to, monitoring higher harmonic frequencies; phaseanalysis or filters; background subtration techniques (including but notlimited to impedance analysis and the use of signal recognition or peakrecognition techniques); digital filtering techniques; bandwidthnarrowing techniques (including lock-in detection schemes particularlydigital lock in); Fast Fourier Transform (FFT) methods; correlationand/or convolution techniques; signal averaging; spectral analysis; etc.Additionally, varying components of the assay can be done to result inthe sample signal and the noise signal being altered in a non-parallelfashion; that is, the two signals respond non-linearly with respect toeach other. These techniques are described in WO00/16089 and O'Connor etal., J. Electroanal. Chem. 466(2):197-202 (1999), hereby expresslyincorporated by reference.

In general, non-specifically bound label probes/ETMs show differences inimpedance (e.g. higher impedances) than when the label probes containingthe ETMs are specifically bound in the correct orientation. In apreferred embodiment, the non-specifically bound material is washedaway, resulting in an effective impedance of infinity. Thus, ACdetection gives several advantages as is generally discussed below,including an increase in sensitivity, and the ability to “filter out”background noise. In particular, changes in impedance (including, forexample, bulk impedance) as between non-specific binding ofETM-containing probes and target-specific assay complex formation may bemonitored.

Accordingly, when using AC initiation and detection methods, thefrequency response of the system changes as a result of the presence ofthe ETM. By “frequency response” herein is meant a modification ofsignals as a result of electron transfer between the electrode and theETM. This modification is different depending on signal frequency. Afrequency response includes AC currents at one or more frequencies,phase shifts, DC offset voltages, faradaic impedance, etc.

Once the assay complex including the target sequence and label probe ismade, a first input electrical signal is then applied to the system,preferably via at least the sample electrode (containing the complexesof the invention) and the counter electrode, to initiate electrontransfer between the electrode and the ETM. Three electrode systems mayalso be used, with the voltage applied to the reference and workingelectrodes. The first input signal comprises at least an AC component.The AC component may be of variable amplitude and frequency. Generally,for use in the present methods, the AC amplitude ranges from about 1 mVto about 1.1V, with from about 10 mV to about 800 mV being preferred,and from about 10 mV to about 500 mV being especially preferred. The ACfrequency ranges from about 0.01 Hz to about 100 MHz, with from about 10Hz to about 10 MHz being preferred, and from about 100 Hz to about 20MHz being especially preferred.

The use of combinations of AC and DC signals gives a variety ofadvantages, including surprising sensitivity and signal maximization.

In a preferred embodiment, the first input signal comprises a DCcomponent and an AC component. That is, a DC offset voltage between theworking and counter electrodes is swept through the electrochemicalpotential of the ETM (for example, when ferrocene is used, the sweep isgenerally from 0 to 500 mV) (or alternatively, the working electrode isgrounded and the counter electrode is swept from 0 to −500 mV). Thesweep is used to identify the DC voltage at which the maximum responseof the system is seen. This is generally at or about the electrochemicalpotential of the ETM. Once this voltage is determined, either a sweep orone or more uniform DC offset voltages may be used. DC offset voltagesof from about −1 V to about +1.1 V are preferred, with from about −500mV to about +800 mV being especially preferred, and from about −300 mVto about 500 mV being particularly preferred. In a preferred embodiment,the DC offset voltage is not zero. On top of the DC offset voltage, anAC signal component of variable amplitude and frequency is applied. Ifthe ETM is present, and can respond to the AC perturbation, an ACcurrent will be produced due to electron transfer between the electrodeand the ETM. These voltages are meaningful numbers for a Ag vs an AgClreference electrode.

Thus, the devices of the invention preferably provide voltage sourcescapable of delivering both AC and DC currents.

For defined systems, it may be sufficient to apply a single input signalto differentiate between the presence and absence of the ETM (i.e. thepresence of the target sequence) nucleic acid. Alternatively, aplurality of input signals are applied. As outlined herein, this maytake a variety of forms, including using multiple frequencies, multipleDC offset voltages, or multiple AC amplitudes, or combinations of any orall of these.

Thus, in a preferred embodiment, multiple DC offset voltages are used,although as outlined above, DC voltage sweeps are preferred. This may bedone at a single frequency, or at two or more frequencies.

In a preferred embodiment, the AC frequency is varied. At differentfrequencies, different molecules respond in different ways. As will beappreciated by those in the art, increasing the frequency generallyincreases the output current. However, when the frequency is greaterthan the rate at which electrons may travel between the electrode andthe ETM, higher frequencies result in a loss or decrease of outputsignal. At some point, the frequency will be greater than the rate ofelectron transfer between the ETM and the electrode, and then the outputsignal will also drop.

In a preferred embodiment, multiple frequencies with a small AC voltageis applied and the fundamental of each is evaluated. Alternatively, apreferred embodiment utilizes several frequencies with a large ACvoltage, and the harmonics of each are evaluated. Similarly, preferredembodiments utilize several frequencies with a large AC voltage wherethe effect of the different frequencies on the system can result in anoutput that is different from the sum of the outputs at individualfrequencies.

In one embodiment, detection utilizes a single measurement of outputsignal at a single frequency. That is, the frequency response of thesystem in the absence of target sequence, and thus the absence of labelprobe containing ETMs, can be previously determined to be very low at aparticular high frequency. Using this information, any response at aparticular frequency, will show the presence of the assay complex. Thatis, any response at a particular frequency is characteristic of theassay complex. Thus, it may only be necessary to use a single inputfrequency, and any changes in frequency response is an indication thatthe ETM is present, and thus that the target sequence is present.

In a preferred embodiment, the input signals and data processing stepsare done to increase the non-linearity of the system: That is, forexample, the ferrocene response reacts non-linearly, producing aharmonic response in the signal above that in the background; thisharmonic signal from AC voltammetry is most likely the result of aharmonic distortion due to the nonlinear response of the electrochemicalcell; see Yap, J. of Electroanalytical Chem. 454:33 (1998); herebyincorporated by reference. Thus, any techniques that increase thisnon-linearity are desirable. In a preferred embodiment, techniques areused to increase the higher harmonic signals; thus, frequency andphase-sensitive lock-in detection is performed at both the fundamentalfrequency of the applied waveform and also at multiples of thefundamental frequency (i.e. the higher harmonics) or just one. Since thebackground capacitance responds relatively linearly to AC signals (asine wave input AC voltage results in a relatively nondistorted sinewave output), very little upper harmonic current is produced in thebackground. This gives a dramatic increase in the signal to noise ratio.Thus, detection at the higher harmonic frequencies, particularly thethird, fourth and fifth harmonics (although the harmonics from second totenth or greater can also be used) is shown to result in dramaticsuppression of the background currents associated with non-Faradaicprocesses (like double layer charging) that can overwhelm the signalfrom the target molecules. In this way, the evaluation of the system athigher harmonic frequencies and phases can lead to significantimprovements in the detection limits and clarity of signal. However, insome embodiments, the analysis of higher harmonics is not desired.

Thus, in a preferred embodiment, one method of increasing the non-linearharmonic response is to increase or vary the amplitude of the ACperturbation, although this may also be used in monitoring thefundamental frequency as well. Without being bound by theory, it appearsthat increasing the amplitude increases the driving force nonlinearly.Thus, generally, the same system gives an improved response (i.e. higheroutput signals) at any single frequency through the use of higheroverpotentials at that frequency. Thus, the amplitude may be increasedat high frequencies to increase the rate of electron transfer throughthe system, resulting in greater sensitivity. In addition, this may beused, for example, to induce responses in slower systems such as thosethat do not possess optimal spacing configurations.

In a preferred embodiment, measurements of the system are taken at leasttwo separate amplitudes or overpotentials, with measurements at aplurality of amplitudes being preferred. As noted above, changes inresponse as a result of changes in amplitude may form the basis ofidentification, calibration and quantification of the system. Inaddition, one or more AC frequencies can be used as well.

In a preferred embodiment, harmonic square wave AC voltage is used; seeBaranski et al., J. Electroanal. Chem. 373:157 (1994), incorporatedherein by reference, although in some embodiments this is not preferred.This gives several potential advantages. For example, square waves areeasier to create digitally and the pulse shape of the square wave canallow for better discrimination against charging capacitance. Insinusoidal harmonic AC voltammetry, harmonic signals provide bettersignal to background since faradaic response can be more nonlinear thancharging capacitance. The same concept applies to SW harmonic ACvoltage. The key difference between the two techniques is the frequencyspectrum of the AC waveform. A singular frequency sinusoidal waveformcontains just the fundamental frequency whereas a singular square wavecontains the fundamental frequency as well as all odd harmonics. Thetechnique looks at the even harmonics where the ratio of faradaiccurrent to capacitance current is enhanced. All the odd harmonics havesingle AC voltage peaks while all the even harmonics have double ACvoltage peaks. This is opposite to the case of sinusoidal harmonic ACvoltage of a system that has a non-reversible redox couple.

In a preferred embodiment, multiple frequency AC voltage is used. Theidea is to create a waveform consisting of multiple frequencies with thesame amplitude or different amplitudes to excite an electrochemical cellin an AC voltage fashion. The method benefits from fast Fouriertransform or joint time-frequency transform to analyze the cellresponse. A JTFT spectrogram of a multiple frequencies AC voltageprovides information on the driven (or fundamental) frequencies as wellas their harmonic components. Some possible data analyses are: 1)comparison of response of fundamental frequencies, 2) comparison of allharmonic frequencies, 3) comparison of the response of one particularharmonic frequency of all excited frequencies, and 4) all analysespossible by standard single frequency AC voltage.

Accordingly, in a preferred embodiment, a fast Fourier transform isdone, as is generally outlined in the examples. Fourier transformanalysis is a preferred method for improving signal to noise andisolating desired signals when sinusoidal electrochemistry is done.Typical AC techniques rely on measurements of the primary frequencyonly. With sinusoidal voltammetry (and other inputs) observation athigher harmonics allows discrimination of signals primarily based onkinetics. For example, both fast and slow redox events would givesimilar peaks (provided the AC frequency was not too high) at theprimary frequency. However, at higher harmonics, some redox moleculeswould generate signals while others would not. Using FFT analysis, allthe various frequency components of a response to a sinusoidal input canbe observed at once.

Similarly, in a preferred embodiment, a joint time-frequency transform(JTFT) is done.

In a preferred embodiment, digital lock-in techniques are used. In thepast, digitized raw data from the electrochemical cell have beenanalyzed by either fast Fourier transform or some complex form of jointtime-frequency transform analysis. The major draw back of these methods,is the enormous computational time associated with frequencytransformation techniques. Digital lock-in, on the other hand, is simpleand fast. In principle, digital lock-in is identical to analog lock-in.In the former case, the bandwidth narrowing process is donemathematically by multiplying the cell response by a sinusoidal with thesame frequency as the input voltage, but with 90_phase shift. Thetechnique has the same limitation as its analog counterpart since onlyone frequency can be analyzed at a time. However, unlike analog lock-in,other frequencies can also be analyzed sequentially (or in parallel witha more powerful processor) since the raw data is archived. For an inputvoltage of

V _(in) =E _(dc) +rt+E _(ac) Sir(ωt)  (1)

the cell's response is essentially

$\begin{matrix}\begin{matrix}{{I(t)} = {\sum\limits_{a}{{I_{n}(v)}{{Sin}\left( {{n\; \omega \; t} - \varphi_{n}} \right)}}}} \\{= {{\sum\limits_{n}{{I_{n}^{\prime}(v)}{{Sin}\left( {n\; \omega \; t} \right)}}} - {{I_{n}^{''}(v)}{{Cos}\left( {n\; \omega \; t} \right)}}}}\end{matrix} & (2)\end{matrix}$

To find the voltage dependent coefficients I_(n) for the frequency (n₀w) we multiply the response by 2 Sin(_n₀t) and −2 Cos(_n₀t) and apply alow pass filter to get the real and imaginary components. The low passfiltering used in this example is a simple moving average.Mathematically, the process is expressed as

$\begin{matrix}{{{\frac{1}{{t\; 1} - {t\; 0}}{\int_{t\; 0}^{t\; 1}{\left( {{\sum\limits_{n}{{I_{n}^{\prime}(v)}{{Sin}\left( {n\; \omega \; t} \right)}}} - {{I_{n}^{''}(v)}{{Cos}\left( {n\; \omega \; t} \right)}}} \right)2\; {{Sin}\left( {{wn}_{0}t} \right)}{t}}}} = {{{\frac{I_{n}^{\prime}(v)}{{t\; 1} - {t\; 0}}\left( {t - \frac{{Sin}\left( {2n_{0}\omega \; t} \right)}{2}} \right)}_{{t\; 1},{t\; 0}}} = {I_{n}^{\prime}(v)}}},{{{{for}\mspace{14mu} t_{1}} - t_{0}}\operatorname{>>}T}} & (3)\end{matrix}$

In a preferred embodiment, background subtraction of the current vectorand phase optimization is done.

In a preferred embodiment, correlation and/or, convolution techniquesare used. In this embodiment, many scans of the same electrode. Ratherthan looking for a peak in a single scan, many scans are viewed and acommon correlation between the scans. For instance, it is possible thata bump in the noise appears near 180 mV for a negative, even if noferrocene is present. However, it is unlikely that the same bump willappear in the same place if the frequencies are scanned. Thus, preferredembodiments take scans at many frequencies and only count a positive ifa peak occurs in all of them. This is a very simple correlation; morecomplex correlations may be done as well.

In a preferred embodiment, signal recovery is done using signalrecognition and background subtraction. In this embodiment, the idea isto fit the cell response to two summed functions, one that describes thesignal and the other that models the background capacitive current. Oncethe functions are constructed, the signal is easily recovered from theresponse by subtracting the fitted background capacitive current. Thissignal recognition scheme is applicable to any system where the signalhas a behavior and shape that is relatively well known. The followingexample illustrates how such a scheme can be applied to the systems ofthe invention.

The response from an electrochemical cell can be processed with alock-in amplifier or equivalent bandwidth-narrowing technique. This isone of many methods of increasing signal to background using some formof bandwidth-narrowing technique.

In a preferred embodiment, spectral analysis of the signal is done. Inthis embodiment, filtering techniques in the frequency domain make useof means, variances, densities, autocorrelation functions, and powerspectral densities of the signal and apply it to the present systems toenhance the signal to noise ratio (see Schwartz et al., SignalProcessing: Discrete Spectral Analysis, Detection, and Estimation, N.Y.McGraw Hill, 1975, hereby incorporated by reference).

In a preferred embodiment, digital filtering techniques are used. Theseinclude, but are not limited to, match filter, Weiner filtering, Kalman,Finite Impulse Response, infinite impulse response, narrow bandfiltering, etc.

In a preferred embodiment, a match filter is used; see Ziemer et al.,“Principles of Communication Systems, Modulation and Noise”, 4th Ed.John Wiley & Sons Inc., New York, 465-471, 1988; and Helstrom, C. W.,“Statistical Theory of Signal Detection”, Pergamon Press, Oxford,112-115, 1968, both of which are incorporated by reference. In itssimplest form, a match filter is a signal processing technique that“weights” the measured response (signal plus noise) samples by somecorresponding known signal amplitude and convolutes the two signals toenhance signal to noise.

In a preferred embodiment, a Weiner filter is used (see Press, supra;and Elliot et al., Fast Transforms: Algorithm, Analysis, ApplicationsN.Y. Academic Press (1982), both of which are incorporated byreference). Weiner filtering involves finding an optimal filter thatremoves noise or background from the “corrupted” signal. This signalprocessing method works in conjunction with Fourier transformtechniques. The idea is as follows. Due to poor signal to noise or alarge background, the output from the instrument is a “corrupted” signal

e(t)=s(t)+n(t)

where s(t) is the signal and n(t) is the noise. Note that s(t) is notthe signal we're after, it is composed of the true uncorrupted signalu(t) convolved with some known response function r(t) (In the case ofthe CMS system with a redox couple, u(t) is the Nemstian). In otherwords,

s(t) = ∫_(−∞)^(∞)r(t − τ)u(τ) τ.

In frequency space, the relation is

S(ω)=R(ω)U(ω),

where S, R, and U are the Fourier transform of s, r, and u,respectively. The uncorrupted signal can be recovered by finding theoptimal filter φ(t) or its Fourier counterpart Φ(ω) which when appliedto the measured signal c(t) or C(ω), and then deconvolved by r(t) orR(ω), produces a signal that approximates the uncorrupted signal u(t) orU(ω) with

${\upsilon (\omega)} = {\frac{{C(\omega)}{\Phi (\omega)}}{R(\omega)}.}$

In general the optimal filter is defined as

${\Phi (\omega)} = {\frac{{{S(\omega)}}^{2}}{{{S(\omega)}}^{2} + {{N(\omega)}}^{2}}.}$

In a preferred embodiment, a kalman filter is used, which is arecursive-estimation filtering technique that tracks the current valueof a changing signal in the presence of noise. See Kalman et al., A NewApproach to Linear Filtering and Prediction Problems, Trans. ASME J.Basic Engineering, Seires D, 82, Mar. 35, 1960; Elliot Ed. Handbook ofDigital Signal Processing: Engineering Applications”, Academic Press,San Diego, p908, 1987; Chui et al., Kalman-Filtering: with Real TimeApplications”, Springer-Verlag, New York, 1987; all of which areexpressly incorporated by reference.

In a preferred embodiment, the non-linear harmonic response is increasedby inducing an asymmetrical response. In a preferred embodiment, this isdone by using a system that has a non-reversible redox couple. Forexample, ferrocene is a redox couple that is very reversible. Thus, theferrocenes subtended by the ac voltage at a given point, get oxidized onthe upswing of the ac voltage and reduced on the down swing. However, Ifa semi-reversible or non-reversible redox couple is used, for example,the molecule will get oxidized on the up swing and not reduced (or aportion) on the downswing; or vice versa. This will produce even greaternon-linearities at certain frequencies.

Three examples of ways to perform this are: use an ETM molecule thatgets degraded in the oxidized form, like luminol, use co-reduction orredox mediation, and use enzyme coupled mediation, as generallydescribed in WO00/16089.

In a preferred embodiment, electron transfer is initiated usingalternating current (AC) methods. In addition, the use of AC techniquesallows the significant reduction of background signals at any singlefrequency due to entities other than the ETMs, i.e. “locking out” or“filtering” unwanted signals. That is, the frequency response of acharge carrier or redox active molecule in solution will be limited byits diffusion coefficient and charge transfer coefficient. Accordingly,at high frequencies, a charge carrier may not diffuse rapidly enough totransfer its charge to the electrode, and/or the charge transferkinetics may not be fast enough. This is particularly significant inembodiments that do not have good monolayers, i.e. have partial orinsufficient monolayers, i.e. where the solvent is accessible to theelectrode. As outlined above, in DC techniques, the presence of “holes”where the electrode is accessible to the solvent can result in solventcharge carriers “short circuiting” the system, i.e. they reach theelectrode and generate background signal. However, using the present ACtechniques, one or more frequencies can be chosen that prevent afrequency response of one or more charge carriers in solution, whetheror not a monolayer is present. This is particularly significant sincemany biological fluids such as blood contain significant amounts ofredox active molecules which can interfere with amperometric detectionmethods.

In a preferred embodiment, measurements of the system are taken at leasttwo separate frequencies, with measurements at a plurality offrequencies being preferred. A plurality of frequencies includes a scan.For example, measuring the output signal, e.g., the AC current, at a lowinput frequency such as 1-20 Hz, and comparing the response to theoutput signal at high frequency such as 10-100 kHz will show a frequencyresponse difference between the presence and absence of the ETM. In apreferred embodiment, the frequency response is determined at least two,preferably at least about five, and more preferably at least about tenfrequencies.

After transmitting the input signal to initiate electron transfer, anoutput signal is received or detected. The presence and magnitude of theoutput signal will depend on a number of factors, including theoverpotential/amplitude of the input signal; the frequency of the inputAC signal; the composition of the intervening medium; the DC offset; theenvironment of the system; the nature of the ETM; the solvent; and thetype and concentration of salt. At a given input signal, the presenceand magnitude of the output signal will depend in general on thepresence or absence of the ETM, the placement and distance of the ETMfrom the surface of the monolayer and the character of the input signal.In some embodiments, it may be possible to distinguish betweennon-specific binding of label probes and the formation of targetspecific assay complexes containing label probes, on the basis ofimpedance.

In a preferred embodiment, the output signal comprises an AC current. Asoutlined above, the magnitude of the output current will depend on anumber of parameters. By varying these parameters, the system may beoptimized in a number of ways.

In general, AC currents generated in the present invention range fromabout 1 femptoamp to about 1 milliamp, with currents from about 50femptoamps to about 100 microamps being preferred, and from about 1picoamp to about 1 microamp being especially preferred.

In a preferred embodiment, the output signal is phase shifted in the ACcomponent relative to the input signal. Without being bound by theory,it appears that the systems of the present invention may be sufficientlyuniform to allow phase-shifting based detection. That is, the complexbiomolecules of the invention through which electron transfer occursreact to the AC input in a homogeneous manner, similar to standardelectronic components, such that a phase shift can be determined. Thismay serve as the basis of detection between the presence and absence ofthe ETM, and/or differences between the presence of target-specificassay complexes comprising label probes and non-specific binding of thelabel probes to the system components.

The output signal is characteristic of the presence of the ETM; that is,the output signal is characteristic of the presence of thetarget-specific assay complex comprising label probes and ETMs. In apreferred embodiment, the basis of the detection is a difference in thefaradaic impedance of the system as a result of the formation of theassay complex. Faradaic impedance is the impedance of the system betweenthe electrode and the ETM. Faradaic impedance is quite different fromthe bulk or dielectric impedance, which is the impedance of the bulksolution between the electrodes. Many factors may change the faradaicimpedance which may not effect the bulk impedance, and vice versa. Thus,the assay complexes comprising the nucleic acids in this system have acertain faradaic impedance, that will depend on the distance between theETM and the electrode, their electronic properties, and the compositionof the intervening medium, among other things. Of importance in themethods of the invention is that the faradaic impedance between the ETMand the electrode is significantly different depending on whether thelabel probes containing the ETMs are specifically or non-specificallybound to the electrode.

Accordingly, the present invention further provides electronic devicesor apparatus for the detection of analytes using the compositions of theinvention. The apparatus includes a test chamber for receiving a samplesolution which has at least a first measuring or sample electrode, and asecond measuring or counter electrode. Three electrode systems are alsouseful. The first and second measuring electrodes are in contact with atest sample receiving region, such that in the presence of a liquid testsample, the two electrophoresis electrodes may be in electrical contact.

In a preferred embodiment, the apparatus also includes detectionelectrodes comprising a single stranded nucleic acid capture probecovalently attached via an attachment linker, and a monolayer comprisingconductive oligomers, such as are described herein.

The apparatus further comprises an AC voltage source electricallyconnected to the test chamber; that is, to the measuring electrodes.Preferably, the AC voltage source is capable of delivering DC offsetvoltage as well.

In a preferred embodiment, the apparatus further comprises a processorcapable of comparing the input signal and the output signal. Theprocessor is coupled to the electrodes and configured to receive anoutput signal, and thus detect the presence of the target nucleic acid.

Once made, the multiplexing devices and cartridges of the invention finduse in a wide variety of applications. In particular, the compositionsof the invention find use in hybridization assays. As will beappreciated by those in the art, electrodes can be made that have asingle species of nucleic acid, i.e. a single nucleic acid sequence, ormultiple nucleic acid species.

Recent focus has been on the analysis of the relationship betweengenetic variation and phenotype by making use of polymorphic DNAmarkers. Previous work utilized short tandem repeats (STRs) aspolymorphic positional markers; however, recent focus is on the use ofsingle nucleotide polymorphisms (SNPs), which occur at an averagefrequency of more than 1 per kilobase in human genomic DNA. Some SNPs,particularly those in and around coding sequences, are likely to be thedirect cause of therapeutically relevant phenotypic variants and/ordisease predisposition. There are a number of well known polymorphismsthat cause clinically important phenotypes; for example, the apoE2/3/4variants are associated with different relative risk of Alzheimer's andother diseases (see Cordor et al., Science 261 (1993)). Multiplex PCRamplification of SNP loci with subsequent hybridization tooligonucleotide arrays has been shown to be an accurate and reliablemethod of simultaneously genotyping at least hundreds of SNPs

The present invention is directed to methods of determining the sequenceof a target nucleic acid at a particular position, using electrochemicaldetection on an electrode. The invention preferably includes thedetection (and optionally quantification) of differences or variationsof sequences (e.g. SNPs) using electrode arrays for detection of thevariation.

As is known in the art, there are a number of techniques that can beused to detect or determine the identity of a base at a particularlocation in a target nucleic acid, including, but not limited to, theuse of temperature, competitive hybridization of perfect and imperfectprobes to the target sequence, sequencing by synthesis, for exampleusing single base extension techniques (sometimes referred to as“minisequencing”), the oligonucleotide ligase amplification (OLA)reaction, rolling circle amplification (RCA), allelic PCR, competitivehybridization and Invader™ technologies. In addition, the presentinvention is directed to a novel invention that capitalizes on novelproperties of surface-bound arrays, and uses “competimers” to reducenon-specific binding.

Thus, the compositions of the present invention may be used in a varietyof research, clinical, quality control, or field testing settings.

In a preferred embodiment, the probes are used in genetic diagnosis. Forexample, probes can be made using the techniques disclosed herein todetect target sequences such as the gene for nonpolyposis colon cancer,the BRCA1 breast cancer gene, P53, which is a gene associated with avariety of cancers, the Apo E4 gene that indicates a greater risk ofAlzheimer's disease, allowing for easy presymptomatic screening ofpatients, mutations in the cystic fibrosis gene, or any of the otherswell known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofanti-HIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-II, may be detected inthis way. Bacterial infections such as tuberculosis, clymidia and othersexually transmitted diseases, may also be detected, for example usingribosomal RNA (rRNA) as the target sequences.

In a preferred embodiment, the nucleic acids of the invention find useas probes for toxic bacteria in the screening of water and food samples.For example, samples may be treated to lyse the bacteria to release itsnucleic acid (particularly rRNA), and then probes designed to recognizebacterial strains, including, but not limited to, such pathogenicstrains as, Salmonella, Campylobacter, Vibrio cholerae, Leishmania,enterotoxic strains of E. coli, and Legionnaire's disease bacteria.Similarly, bioremediation strategies may be evaluated using thecompositions of the invention.

In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

In an additional embodiment, the probes in an array are used forsequencing by hybridization.

Thus, the present invention provides for extremely specific andsensitive probes, which may, in some embodiments, detect targetsequences without removal of unhybridized probe. This will be useful inthe generation of automated gene probe assays.

Alternatively, the compositions of the invention are useful to detectsuccessful gene amplification in PCR, thus allowing successful PCRreactions to be an indication of the presence or absence of a targetsequence. PCR may be used in this manner in several ways. For example,in one embodiment, the PCR reaction is done as is known in the art, andthen added to a composition of the invention comprising the targetnucleic acid with a ETM, covalently attached to an electrode via aconductive oligomer with subsequent detection of the target sequence.Alternatively, PCR is done using nucleotides labelled with a ETM, eitherin the presence of, or with subsequent addition to, an electrode with aconductive oligomer and a target nucleic acid. Binding of the PCRproduct containing ETMs to the electrode composition will allowdetection via electron transfer. Finally, the nucleic acid attached tothe electrode via a conductive polymer may be one PCR primer, withaddition of a second primer labelled with an ETM. Elongation results indouble stranded nucleic acid with a ETM and electrode covalentlyattached. In this way, the present invention is used for PCR detectionof target sequences.

In a preferred embodiment, the arrays are used for mRNA detection. Apreferred embodiment utilizes either capture probes or capture extenderprobes that hybridize close to the 3′ polyadenylation tail of the mRNAs.This allows the use of one species of target binding probe fordetection, i.e. the probe contains a poly-T portion that will bind tothe poly-A tail of the mRNA target. Generally, the probe will contain asecond portion, preferably non-poly-T, that will bind to the detectionprobe (or other probe). This allows one target-binding probe to be made,and thus decreases the amount of different probe synthesis that is done.

In a preferred embodiment, the use of restriction enzymes and ligationmethods allows the creation of “universal” arrays. In this embodiment,monolayers comprising capture probes that comprise restrictionendonuclease ends, as is generally depicted in FIG. 7 of PCT US97/20014.By utilizing complementary portions of nucleic acid, while leaving“sticky ends”, an array comprising any number of restrictionendonuclease sites is made. Treating a target sample with one or more ofthese restriction endonucleases allows the targets to bind to the array.This can be done without knowing the sequence of the target. The targetsequences can be ligated, as desired, using standard methods such asligases, and the target sequence detected, using either standard labelsor the methods of the invention.

The present invention provides methods which can result in sensitivedetection of nucleic acids. In a preferred embodiment, less than about10×10⁶ molecules are detected, with less than about 10×10⁵ beingpreferred, less than 10×10⁴ being particularly preferred, less thanabout 10×10³ being especially preferred, and less than about 10×10²being most preferred. As will be appreciated by those in the art, thisassumes a 1:1 correlation between target sequences and reportermolecules; if more than one reporter molecule (i.e. electron transfermoeity) is used for each target sequence, the sensitivity will go up.

All references cited herein, including all patent applications areincorporated by reference in their entirety.

EXAMPLES Example 1 Signal Analysis

The present invention utilizes electrochemical techniques to detectvarious biological and chemical targets. Generally these techniquesyield signals with an informative or a characteristic shape, size, andlocation. By creating a computer program that recognizes a signal'scharacteristic features, we can distinguish signals from backgroundphenomena and extract any relevant information necessary for accuratedetection.

Fitting to a Model

It's generally possible to design a family of equations and a set ofboundary conditions that describe the signals that can arise from agiven measurement technique. This mathematical description is called a“model.” Sometimes the model is based on underlying scientific theory,but in many cases it may simply be an approximation that matches theobserved signal behavior. In most cases the model is “non-linear,”comprising equations that are more complicated than basic polynomials.

There are several ways to fit data to a non-linear model, but theycommonly involve the following steps: 1) the rapid detection of anycommon behaviors not described by the model, 2) an initial guess, 3)iterative improvement and evaluation, repeated as necessary, 4) thedetection and correction of common erroneous fits, if any, and 5) afinal evaluation to judge the quality of the fit. Once a fit is chosen,the values of important parameters can be extracted for use in furtherdata analysis.

Vector Notation for Describing AC Signals

As an example illustrating the signal recognition methods described inthis report, I will use what is currently our most commonelectrochemical technique: Alternating Current (AC) Voltammetrymonitored at the fourth harmonic. This technique yields an AC signal (asine wave) that varies its amplitude (height, R) and phase (position,(θ) as a function of the input DC voltage. As long as we monitor at aknown frequency, it only takes two values to define such a wave. FIG. 16depicts a sine wave and its corresponding vector notation.

The two values can be R and θ, but as shown in the figure they can alsobe an (X,Y) pair separated by one quarter of an oscillation, i.e. by90°. One way to simplify the visualization of such a system is by usingwhat is called vector notation, demonstrated in four configurations inFIG. 17.

It's important to observe that the values (R,θ) and (X,Y) are differentbut interchangeable ways of describing the same vector. The vectoritself is what represents the sine wave and, therefore, the data.Furthermore, the difference between the primed and unprimed values(those on the right side of the diagram versus those on the left) isonly a rotated frame of reference (as indicated, for example, by therelative positions of the dotted lines in the polar coordinatediagrams). This rotation also does not alter the data, but can be usefulas described in later sections.

One important attribute of vector notation is that the vectors addexactly like the corresponding waves. For example, if two vectors pointin roughly opposite directions, when they add they tend to cancel oneanother, leaving only a small residual vector. This exactly models howit is possible to add two waves together in such a way as to have“destructive interference,” where the resulting amplitude is less thaneach of the inputs. As long as all waves have the same frequency,vectors will model their interference with one another.

In AC Voltammetry, we monitor the oscillations at a given frequency as afunction of an input voltage. Since vector notation exactly modelssinusoidal behavior at any single, known frequency, in my description ofour fitting I will only describe the data as a vector.

Choosing to Fit in X and Y

During any experiment acquiring vectoral data, it is common forscientists to only actively monitor the value of R (even if both R and θare recorded). This is because, depending on the system and experimentalsetup, the frame of reference may change from one instrument to anotheror from one day to the next. R, however, does not change with the frameof reference. (Remember that, in polar coordinates, a frame of referencerotation only changes θ.)

However, in order for signal recognition to work we need a model, amathematical description of the shapes we expect to observe. The simplerand less varied the shape, the easier the description and recognition.FIGS. 18 and 19 are examples of R and θ traces for fourth harmonic ACvoltammetry (AC voltage-4).

The four-lobed shape in R-space is characteristic of medium to largesignals, but as the signal shrinks relative to the size of thebackground, the R-space signal distorts. Furthermore, e traces of scanswith larger signals are quite different from those with smaller ones.FIGS. 20 and 21 depict examples of a smaller signal.

This complex (R,θ) behavior is a characteristic of vectoral traces thatcomprise both signal and background. If we have signal S (described byR_(S) and θ_(S)) with a background B (described by R_(B) and θ_(B)),then the data is D= S+ B. D is described by R_(D) and θ_(D), which havedependence on the signal and background values:

$R_{D} = \sqrt{\left( {{R_{S}\sin \; \theta_{S}} + {R_{B}\sin \; \theta_{B}}} \right)^{2} + \left( {{R_{S}\cos \; \theta_{S}} + {R_{B}\cos \; \theta_{B}}} \right)^{2}}$$\theta_{D} = {\arctan \left( \frac{{R_{S}\sin \; \theta_{S}}\; + {R_{B}\sin \; \theta_{B}}}{{R_{S}\cos \; \theta_{S}} + {R_{B}\cos \; \theta_{B}}} \right)}$

The complexity of R and θ traces comes from the fact that R_(D) dependson all four parameters (R_(S), θ_(S), R_(B), and θ_(B)), as does θ_(D).However, if we use Cartesian coordinates to describe the data instead ofpolar ones, then D is described by X_(D) and Y_(D), which are:

X _(D) =X _(S) +X _(B)

and

Y _(D) =Y _(S) +Y _(B)

Using Cartesian coordinates simplifies the dependence of D's parameterson those of S and B. This simplicity is exhibited when graphing the sameexamples shown previously, but now as (X,Y), as depicted in FIGS. 22 and23 (medium sized signal), and FIGS. 24 and 25 (smaller signal).

The smaller signal is now qualitatively similar to the medium signal,and is therefore more likely to be described by the same mathematicalmodel. Because of this, we chose to fit in X and Y. (For simplicity inconceiving a model and in computation during fitting, we chose to fitindependently in X and Y instead of fitting both dimensionssimultaneously.)

The Model Assumed for AC voltage-4

We have compared the characteristic shape exhibited by AC voltage-4signals to several different mathematical expressions. The four-lobedprofile immediately suggested we use an equation related to the thirdderivative of a peak shape, and after making many comparisons, weconcluded that the third derivative of a Gaussian (G′″) was a very goodapproximation to an AC voltage-4 signal. As for the background, apolynomial (P) should be sufficient to account for the majority of thedifferent shapes that we observe. (The order of polynomial (andtherefore the number of parameters needed to describe its shape) willdepend on the length of the scan. Longer scans will require higher orderpolynomials to account for the same scan features.) This translates tothe following equations, where Ī(V) is the data, I_(S) (V) is thesignal, and I_(B) (V) is the background:

I (V)= I _(S) (V)+ I _(B) (V)=└ G _(X)′″(V)+ G _(Y)′″(V)┘+ P _(X) (V)+ P_(r) (V)┘=└ G _(X)′″(V)+ P _(X) (V)┘+ G _(r)′″(V)+ P _(r) (V)┘

This leads to the final equations we used for our AC voltage-4 model:

X(V)=G _(X)′″(V)+P _(X)(V)

and

Y(V)=G _(Y)′″(V)+P _(Y)(V)

We have also created fitting procedures for systems with more than onelabel (differentiated by their position in voltage space). They use amodel analogous to that described above, but with the underlyingassumption of more than one signal, that is:

${\overset{\_}{I}(V)} = {{\overset{\_}{I_{B}}(V)} + {\sum\limits_{n}{\overset{\_}{I_{S,n}}(V)}}}$

As for boundary conditions, note that the Gaussian derivatives G_(X)′″and G_(Y)′″ each have three parameters: one for height, one for width,and one for location (in voltage). The height has no restrictions,corresponding only to the number of electrochemical labels that aresignaling. However, in order to represent a true electrochemical signal,a fit's width must fall within a reasonable range. Furthermore, signalsin the independent X and Y fits must be close to one another (in voltagespace) to assure that they both correspond to the same electrochemicallabel. Later I will discuss how these boundary conditions may be“enforced” to assure a meaningful fit.

Optimal Phase

As mentioned previously, the choice of reference frame is arbitrary. Asfar as the data is concerned, one (X,Y) pair is just as good as another,rotated (X′,Y′) pair. However, since the model is often an approximationto reality instead of an exact theoretical description, the model mayimpose a preferred frame of reference. This is true of the AC voltage-4system described above.

As an example, lets consider one AC voltage-4 trace that has a largesignal relative to the background. If, in a two-dimensional graph, weplot the tip of the data vector as a function of voltage (one point isplotted every 10 mV), it results in FIG. 26.

If we choose a frame of reference such that the X and Y axes straddlethe signal, as shown above, then the signal contributes strongly to bothX and Y, as shown in FIGS. 27 and 28.

However, if we choose an axis pair that is roughly parallel andperpendicular to the signal (rotated 45

with respect to the axes drawn in FIG. 26), very little of the signalcontributes to the perpendicular vector as shown in FIGS. 29 and 30.

Furthermore, we can see that the four-lobed shape we chose for the ACvoltage-4 model does not describe the six lobes of the perpendiculartrace. If we were to try to use the model to fit the data using theparallel and perpendicular (X,Y) pair, we would only be able to extractthe signal out of the parallel component, thus losing one of thedimensions of our data.

Instruments generally assign the X and Y axes based on the phase of theAC input driving force. Because this choice does not take into accountthe electrochemical system, it's possible that it may lead to theparallel/perpendicular trouble described above. Therefore, for signalrecognition based on the above model, it's best to choose a new pair ofaxes assured to straddle any existing electrochemical signal.

In order to choose such axes, we need a way to measure the signal'sdirection. We could fit a line to the signal in polar coordinates, butwe can't use basic linear fitting since the X and Y signals areindependent of one another. For example, imagine a signal aligned alongthe Y axis. If we attempt a linear, least squares fit (the most commontype of fitting), the resulting line is not along the signal but ratheralong the X axis, with equal number of points above and below thefitting line. This is because the data, when considered only as thevalues in the Y direction, has no X dependence.

A non-linear fit would work, but would be an iterative procedure and sowould take more processing time than we'd care to use. Instead, we'drather use a faster way involving simpler mathematical operations. Onesuch way is using a vectoral sum. Consider the grouping of three pointsshown in FIG. 31.

If we consider these points as vectors, we can add them by summing theircoordinates. The vectoral sum will have coordinates

$X = {\sum\limits_{i}x_{l}}$ and $Y = {\sum\limits_{i}y_{i}}$

This summation of the vectors provides a reasonable angle for the bestline through the data that passes through (0,0).

$\theta_{apr} = {{\arctan \left( \frac{Y}{X} \right)} = {\arctan \left( \frac{\sum\limits_{i}\; y_{i}}{\sum\limits_{i}\; x_{i}} \right)}}$

We call this angle the “optimal phase.” For our example, the summationis drawn in FIG. 32. FIG. 33 shows how the three sample data pointscluster around the line. For them, the optimal phase is 64

.

An advantage to this method is that the results are weighted by thelength of the vectors of the original data points. That is, if a datapoint has a small amplitude (as it will if it represents a segment of ascan where no signal exists), it has a smaller impact on the value ofthe optimal phase. For example, if we add a small data point to thesample grouping, the results are shown in FIGS. 34 and 35. The new pointchanges the optimal phase by less than three degrees.

If desired, it is possible to give the small values even less weight. Amore generic expression for the optimal phase has its summationsweighted by the lengths (r_(i)) of the individual data points' vectors.Increasing the value of n places less and less emphasis on the smalldata points. However, in all of our current fitting programs, we use theequation as written above, equivalent to the case where n remains zero.

$\theta_{apr} = {{\arctan \left( \frac{\sum\limits_{i}\; {y_{i}r_{i}^{*}}}{\sum\limits_{i}\; {x_{i}r_{i}^{*}}} \right)} = {\arctan \left( \frac{\sum\limits_{i}\; {y_{i}\left( {x_{i}^{2} + y_{i}^{2}} \right)}^{n/2}}{\sum\limits_{i}\; {x_{i}\left( {x_{i}^{2} + y_{i}^{2}} \right)}^{n/2}} \right)}}$

We can use this method to calculate the optimal phase for fitting asignal, but there are complications that must be considered. For manytechniques (including AC voltage-4), the electrochemical signal isshaped such that portions of it may cancel each other out whencompleting the calculation described above. To avoid this, we firstrotate one half of the data 180

. Taking the data shown in FIG. 26, we calculate the optimal phase usingthe data as shown in FIG. 36. The resulting line is overlaid on theoriginal data, at FIG. 37.

The angle of the line drawn in FIGS. 36 and 37 (101

) is what was used to choose the X and Y axes (at ±45

) for this file. Unfortunately, however, there can be a furthercomplication. If the signal is oriented differently relative to thedividing line between rotated and unrotated segments, the statedmanipulation may not yield the proper angle. For example, if I take theabove signal and rotate it 101 degrees clockwise, its optimal phaseshould be 0

. However, the calculated value actually ends up as −48

as shown in FIG. 38.

To prevent this, we need to choose a rotation boundary that is moreperpendicular to the signal than it is parallel. We can do so by firstdetermining whether the signal lies mostly along 0 degrees or mostlyalong 90. If we take the vectoral sum of the absolute value of thecoordinates of a signal that's closer to 90, the resulting angle

$\arctan \left( \frac{\sum\limits_{i}\; \left| y_{i} \right|}{\sum\limits_{i}\; \left| x_{i} \right|} \right)$

will be greater than 45

. On the other hand, for the above case we find an angle of 10 degrees(see FIG. 39), less than 45, and conclude that the signal is more along0 degrees. Therefore, we rotate the half of the signal from the far sideof the 90 degree axis (see FIG. 40). Calculating the vectoral sum nowyields a reasonable value for the optimal phase: 1

similar to the expected 0

.

One final complication is the fact that we don't want to find theoptimal phase for an entire scan, but rather for any electrochemicalsignal present in a scan. We want to ignore any background.Unfortunately, if the signal is small, the optimal phase calculationsoutlined above would be dominated by the phase of the background. Toavoid this, we perform an approximate background subtraction beforecalculating the vectoral sum.

For example, if we examine the scan represented by FIGS. 24 and 25, wesee a large contribution by the background. If we examine the scan intwo dimensions, we can see that the phase of the entire scan is mostlyalong 120

(FIG. 41).

However, if we perform the rapid calculations necessary to fitpolynomials to the entire scan (one each along the 0 and 90

axes), we can approximate the background as shown in FIGS. 42 and 43. Inthis instance, we are using the symmetry.of the AC voltage-4 to ouradvantage. If a signal has equal contributions above and below thebackground (as is true of any even harmonic AC voltage), then apolynomial fit will tend to follow the centerline of that signal. Thismakes the polynomial fit to the scan an excellent estimation of thebackground. To calculate the polynomial, we currently use a “generalpolynomial singular value decomposition fit”.

This approximation to the background can then be subtracted, convertingthe scan into something that is much more purely signal, as shown inFIGS. 44 and 45. FIG. 46 depicts this as a two dimensional plot, fromwhich we can see that now, using the techniques outlined earlier in thissection, we could calculate an optimal phase of approximately 70

.

In summary, the basic procedure for choosing the X and Y axes is asfollows:

1. Fit an approximate background along each of the 0 and 90

directions. Subtract them from the scan, leaving a residual tracedominated by signal.2. Take the absolute value of all coordinates (along 0 and 90°).Calculate a vectoral sum. Determine if its angle (θ_(abs)) is closer to0° or 90°.3. If θ_(abs) is closer to 0°, select all data points to the left of the90° axis. If closer to 90°, take all data points below the 0° axis.Rotate these 180°. (For simplicity in calculation, we have used the 0°and 90° axes as our boundaries. If a more accurate determination ofθ_(opt) is required, we can instead rotate the set of points that fallsoutside the span of θ_(abs)±90°).4. Calculate a vectoral sum. Its angle is the ‘optimal phase’ (θ_(opt)).5. Choose the X and Y axes at ±45° from θ_(opt).

Checking for Behaviors Not Modeled

To reduce total processing time, it's best to notice early if a scan hasany gross deviations from the model that would make fitting itmeaningless. One such feature we have encountered in AC voltammetry(fourth harmonic) has been the sharp peak caused by the stripping of ametallic contaminant. FIG. 47 shows an example of one displayed inR-space.

In X and Y (at +45

from the optimal phase), the sharp spike feature remains clear, as shownin FIGS. 48 and 49.

The symmetry of this feature distinguishes it from our normal signal:although AC voltage-4 signals have equal portions above and below thebackground, this spike does not.

One quick but rough method of monitoring this symmetry is to separateout an approximate background (as we had done to determine the optimalphase) and then compare the distribution of points above the baselinewith the distribution below. For example, if we subtract a polynomialfrom the Y trace above, we get the results shown in FIGS. 50 and 51.

If we now examine the distribution of data above and below theapproximated background, we find that the presence of the spike causes alarger range of values to exist below the background line than above it,as shown in FIG. 52.

In this example, the standard deviation of the data below the line isabout 2½ times larger than the standard deviation above. We can takethis as an indication of symmetry different from that expected of an ACvoltage-4 signal, since AC voltage-4 signals are distributed evenlyabout the assumed background and therefore have ratios closer to 1. Bysetting a range of acceptable values, this rough method allows for therapid detection and rejection of scans with large spikes. Currently, weconsider a ratio less than 2.25 and greater than 1/2.25 to be acceptablefor further fitting. However, it is important to be aware that thisvalue can depend on unusual parameter, such as scan length.

The Initial Guess

Iterative fitting procedures all require a starting point, an initialguess for the values of the model's equations that would match the data.Iterations (discussed in the following section) then improve upon theseguesses in gradual steps. For systems with simple models, there is oftenonly a need for a single, predetermined initial guess. In that case, theguess is an adequate starting point for all possible data. However, formodels that comprise complex shapes (such as the model we use for ACvoltage-4), an accurate initial guess based on each individual scan canlead to more rapid fitting and can reduce or eliminate any tendency tocreate erroneous fits.

Again, we can use symmetry to our advantage. First, for a signal withsymmetry such that it's distributed equally above and below thebaseline, we can use a fast polynomial fit to calculate an initial guessfor the background. As was true in the optimal phase calculationoutlined in a previous section, this polynomial will tend to fit to thecenterline of any signal, thus making a good background estimation. Animprovement to get better background estimation is down by “nailingdown” the edges, thereby reducing edge effects.

When we subtract out the estimation of the background, what remains(“the residual”) should be mostly signal (if any). To estimate thatsignal, we can begin with the fact that a given measurement techniqueand chemical system generally yields signals with characteristicbehaviors. For sensors probed with AC voltage-4, all signals havesimilar widths. An initial guess based on the most common width istherefore appropriate.

To guess the remaining parameters of signal position and signal height,we can again use the known AC voltage-4 symmetry, this time combinedwith knowledge of the characteristic width. Since we know the averageseparation between the two larger center lobes, we can duplicate thesignal and shift the two copies in opposite directions for half of thatseparation. If we then subtract one from the other, the center lobesinterfere constructively. The absolute value of this resulting waveprovides a good estimation of the height and position of the signal.This process is shown in FIGS. 53, 54 and 55 for a signal 11.9 tall at aposition of 0.20 with a center lobe separation of 0.072.

The trace in FIG. 55 has its largest value, 23.25, at a position of0.20. The position matches well with the true data value. (Both are0.20.) The constructive interference height should be about twice thecenter-to-peak signal height, which means the interference plot gives aninitial guess of ½×23.25=11.6. This is only 3% different from the inputvalue. (This difference occurs because the shift used in the aboveprocess was 0.062, different from the actual 0.072 separation of thesample signal.)

One important advantage of the above method (rather than if we were tosimply search for maxima and minima in the raw data) is that itamplifies only those features with the expected width and symmetry ofthe signal.

Consider the same signal as above, but this time with an unusual peakoff to one side that's slightly taller than the signal itself. (See FIG.56.) In a simple maxima/minima search, this would be likely to interferewith the initial guess. However, using the procedure outlined above, theinitial guess will remain 11.6 tall at a position of 0.20, as shown inFIG. 57.

We therefore have a method of calculating an initial guess for allparameters (the signal height, position, and width, plus the background)that involves only 1) calculating a polynomial using a rapid (linear)fitting method, 2) searching for the largest number in a set, and 3)simple arithmetic. As an example of the power of this technique, FIG. 58is the overlay of a real data trace and the corresponding initial guess.

Optimization and Dynamic Range

Once an initial guess has been made, we can use any of a number ofstandard non-linear regression algorithms to optimize the fit. (Wecurrently use a version based on the Levenberg-Marquardt method. This isdiscussed in Chapter 15 of Numerical Recipes in C: The Art of ScientificComputing, second edition, by W. H. Press, S. A. Teukolsky, W. T.Vetterling, and B. P. Flannery, Cambridge University Press, New York(1992). It was originally presented by D. W. Marquardt as the “maximumneighborhood” method in Journal of the Society for Industrial andApplied Mathematics, 11 (1963) 431-441.) While they differ in thedetails, the basic procedures are all the same. The initial guess iscompared with the actual data, and the fit is altered based on thiscomparison in an attempt to minimize the “error”. We iterate (repeatthis process continuously) until the successive reductions in the errorare smaller than the “precision” (a pre-set constant). In this case, thefit is said to have “converged”. However, if the data doesn't match theentire shape described by the model, it's possible that the alterationsto the fit won't reduce the error. (This will happen, for example, ifthe scan contains neither a signal nor noise that looks like a signal.)If the error doesn't become smaller than the precision within the“maximum number of iterations” (also a pre-set constant), the fit issaid to have “diverged”.

The error and precision are often defined such that their values haveunits. For example, if the data is a current measured in picoamps, thenthe error and precision are in picoamps squared. This is most usefulwhen all expected signals are of similar size, because it considerseverything in absolute terms and will not attempt to optimize fits tosmall features. However, quantitative analytical techniques generallyrequire a wide dynamic range. For example, one may need to examinesignals that are two picoamps tall with the same ease as one examinestwo billion picoamp signals. To achieve such a dynamic range, wenormalize data to the initial guess for the signal height. This allowsthe small signals to be fit just as well as the large, with only theshape and the background noise affecting the fit.

Boundary Conditions and Weighting

The question of boundary conditions arose earlier when discussing thechoice of a model. For some systems, certain parameters may bereasonable only within a certain range. For example, for AC voltage-4the width of the center two lobes of a sensor signal always fallsbetween 110 and 265 mV, and is most commonly between 150 and 200 mV.There are several ways to enforce these boundaries, two of which arediscussed here.

If the initial guess for a scan is fairly accurate and the scan's signalis sizeable relative to the background, the fitting procedure willgenerally lock into the signal properly. There will be no need toenforce the boundary conditions. However, if there's no signal or thesignal is obscured, it's possible for parameters to drift outside oftheir acceptable ranges. Now, for a well-behaved system, the only fitsthat exhibit this drift have no true signals. We can therefore discardfits with unacceptable values and consider the corresponding scans to becomprised only of background. (See “Quantitation of Negatives” below.)This is the procedure most widely used in our current software.

However, for systems that are less well-behaved, we may prefer toenforce the boundary conditions during the fitting itself rather thanafter the fact. We can do this by adding an additional term to theequation that describes the error for each constrained parameter,penalizing the fit as its parameters deviate from the desired range. Forexample, if E is the error as defined by the non-linear regressiontechnique, and if the parameter a is to be constrained to fall close tosome expected value ā, then we can replace E with E′, where k is someconstant and n is an integer:

E′=E+k(a−ā)^(2n)

Remember that, during the iterative optimization, the goal is tominimize the error. If we use the equation above, then the farther a isfrom ā the larger the error becomes and therefore the less favorable thefit. We can use the value of k to determine how important it is toconstrain the parameter relative to the standard error E (and alsorelative to any other parameters' constraints). The value of n affectshow unfavorable a certain range of values is. For example, in the graphin FIG. 59 we compare the shape when the added term has 2n=16 with when2n=2. In the case where 2n=16, a values within ±7 of the expected areall equally acceptable, with little added penalty. However, with 2n=2,there's an increasingly harsh penalty the further a moves from theexpected value.

It's important to note that there's nothing critical about the form ofthe added term, so long as it's always positive. For example, if wewanted a sharper constraint on a near its true value but not so muchdependence when far off, we could use an expression like:

$E^{\prime} = {E + {k_{i}\left\lbrack {\arctan \left( \frac{a - \overset{\_}{a}}{c} \right)} \right\rbrack}^{2}}$

This equation leads to shapes like the ones depicted in FIG. 60.

Even more complicated shapes may be used. For example, for the case ofour AC voltage-4 center lobe pairs (described previously), we may wishto use the shapes shown in FIG. 61, which may be defined by:

${\sum\limits_{i = 1}^{n}\; {b_{i}\left\{ {I + {\tanh \left\lbrack {c_{i}\left( {a - s_{i}} \right)} \right\rbrack}} \right\}}} + {\sum\limits_{j = 1}^{m}\; {b_{j + n}\left\{ {I - {\tanh \left\lbrack {c_{j + n}\left( {a - s_{j + n}} \right)} \right\rbrack}} \right\}}}$

where there are n steps up; m steps down, the s's are the locations ofthe steps, the b's are their heights, and the c's control theirsteepness.

There is another way to use the error to control where various segmentsof a fit lock into a scan. The mathematical definition of the error E(often called the mean square error, or MSE) is below:

$E = {\frac{1}{N}{\sum\limits_{i = 1}^{N}\; \frac{\left( {F_{i} - D_{i}} \right)^{2}}{\sigma_{i}^{2}}}}$

In this equation, N is the number of data points, F_(i) is the value ofthe fit for data point number i, D_(i) is the corresponding data value,and σ's is the standard deviation in this data value. This standarddeviation is a measure of the uncertainty in D_(i), and is generallyknown only if several different measurements were averaged to createD_(i). (For data that has not been averaged, or whose averaginginformation has been lost, a value of σ's=1 is assumed, saying that allvalues are known with equal certainty.) By dividing by σ's, we're sayingthat a larger fitting error is acceptable for a given point if that datavalue was uncertain in the first place.

Irrespective of any uncertainty in data values, we can use the same kindof manipulation to force the fit to match the data more closely incertain data ranges than in others by introducing a weighting w_(i) foreach point. Data points with larger w_(i ′)s (a heavier weighting) willbe fit more closely than those with smaller w_(i)′s:

$E_{urlghard} = {\frac{1}{N}{\sum\limits_{i = 1}^{N}\; {w_{i}^{2}\frac{\left( {F_{i} - D_{i}} \right)^{2}}{\sigma_{i}^{2}}}}}$

(Note that this is exactly the same as introducing an alternate standarddeviation equal to σ_(i)/w_(i).) Now, this does not directly force thesignal portion of the fit to exist in a region with large weighting. Itonly forces the fit as a whole to be tighter in this region relative tothe other segments of the scan. However, the equations that describesignal are more localized than those that describe background, and theygenerally can exhibit much more curvature. Because of this, puttinglarger weightings w_(i) around the expected signal position tends toforce the signal to lock in near that value.

Since the initial guess is such an important factor when calculating agood fit, we need to use the weighting in that step as well. Toaccomplish this, we can multiply the |Shift and Subtract| valuedescribed in the “Initial Guess” section by some function, either basedon the weightings w_(i) or on the term added to create E′, depending onthe method.

Detecting and Correcting Flawed Fits

After completing the optimization and converging to a fit, it's stillpossible for that fit to have locked in improperly. For example, therecan be edge effects. That is, since the computer has no data outside thescan range, it is completely free to assume any shape for the dataoutside that range. Because of this, the fitting procedure may concludethat an unusual background oscillation at the scan's edge is actually asignal. This kind of fit needs to be discarded, or perhaps avoided byusing the weightings described above.

Other possible errors may be corrected. For example, in AC voltage-4 wefit in X and Y independently. Because of this, for small signals or forscans with wavy backgrounds, it's possible for the program to lock intoincorrect lobes of the signal in either X or Y. That is, the fit mayname a satellite and a central peak as the two central peaks. This willmanifest itself as a fit to signal positions that are substantiallydifferent in X and Y. In these cases, we can take the scan (X or Y) thatlocked in too far from the expected position and refit. We base a newinitial guess on the incorrect fit (keeping the background and thesignal width, but correcting the signal position) and restart theiterative optimization. If this does not remedy the problem (or if theoriginal difference in position is very large), then generally it meansthat we've locked into noise rather than a signal, so we discard thisfit.

Judgment of Fit Suitability

Once a fit has converged and those fits with common flaws have beendiscarded or corrected, we need to judge if the fit has too much error.That is, we need to make sure that we've locked into a real signal. Forexample, examine the graph shown in FIG. 62.

Although the fit may closely follow the average path of the data, in theabove case the fit isn't reliable because the difference between the fitand the data is too large. There is too much noise in the scan. We canjudge this quantitatively by setting a threshold value for an acceptableerror E. In the case of AC voltage-4, since we fit in X and Yindependently, we set a threshold value for maximum noise allowable inR-space:

E _(R)=√{square root over (E _(X) ² +E _(Y) ²)}

E_(R) must be less than some empirically-determined value in order forthe fit to be considered as having locked into a true signal.

Quantitation of Negatives

Once all of the above procedures have been completed, many scans havebad fits diverge or be discarded. These are classified as having noobservable signal, and so are refit using only background as the model.For example, FIG. 63 is the R composite, √{square root over (X²+Y²)}. InFIG. 64 is the R composite with the background polynomials subtracted,

$\sqrt{\left( {X - X_{background}} \right)^{2} + \left( {Y - Y_{background}} \right)^{2}}$

After the background has been fit, it's often desirable to extract aquantitative estimation of the size of the largest signal that may behidden within the residual noise. Because the software recognizes shape,all we need to consider are oscillations in the noise that are similarto the shape of the modeled signals. (In AC voltage-4, signals have acharacteristic period in voltage space: the center lobes commonlycomplete one cycle in about 0.16 volts.) By subtracting out thebackground fit and examining only the residual, we remove the lowfrequency background. To remove the high frequency noise, (thusconsidering only the signal-like oscillations), we push the residualthrough a low pass filter. To determine the appropriate filterparameters for AC voltage-4, we averaged the power spectra of the X andY traces of several thousand files with signals. This yielded an averagefrequency (V⁻¹) profile which we used to choose the appropriateparameters to pass all signals. We commonly use an IFIR low pass filter.(The Interpolated FIR filter is described by Y. N. Neuvo et al. in IEEETrans. Acoust., Speech, Signal Processing, vol. ASSP-32, pp. 563-570,June 1984.). Any scan passed through this filter now holds only thoseoscillations that might represent an obscured signal. FIG. 65 shows thefiltered, signal-like noise is drawn on top of the residual (Raw) fromthe previous example.

For AC voltage-4, since the vectoral signal is being fit independentlyin X and Y, the filtered residual can be quantified as follows:

${{filtered}\; R_{{center}\text{-}{to}\text{-}{peak}}} = {{\sqrt{2}\left( {{filtered}\mspace{14mu} R_{RMS}} \right)} = \sqrt{2\left( {{filteredX}_{RMS}^{2} + {filteredY}_{RMS}^{2}} \right)}}$

The RMS measurement was multiplied by sqrt[2] to convert it to acenter-to-peak measure. We named this residual current value i_(r).

i_(r) gives an estimate of how much signal-like noise exists across thescan, but does not account for 1) any attenuation due to the filter, 2)for the fact that our signals are localized in a single region of thescan rather than spread across its entire length, or 3) for any possiblelimitations of the non-linear optimization when extracting a signal fromnoise. Because of this, i_(r) alone would underestimate the largestpossible hidden signal. Therefore we include a multiplicative factor “C”such that C*i_(r) is equal to the largest peak height (i_(p)) that mightbe obscured. (To find the value of C, we calculated i_(p)/i_(r) forseveral thousand files from many different experiments. C was set equalto the value of i_(p)/i_(r) at which the signals disappear.) In theabove example, the largest possible missed signal is C*i_(r)=7.57×10⁻¹².

Background Subtraction and Information Extraction

For scans with signals, once we have a fit, we're armed with all theinformation necessary for data analysis. Using the model as a guide, wecan use the fit parameters to calculate the equation for the backgroundalone and subtract this from the data. For example, in AC voltage-4 wecan subtract the polynomial in X and in Y. FIG. 66 is the original data,FIG. 67 is the data with the background subtracted.

We can also calculate values of interest. For example, for AC voltage-4we can calculate the peak height i, and the peak position E₀ based onthe values fit in X and Y:

$i_{p} = \sqrt{i_{pX}^{2} + i_{pY}^{2}}$$E_{0} = {\frac{{E_{0\; X}i_{pX}^{2}} + {E_{0\; Y}i_{pY}^{2}}}{i_{pX}^{2} + i_{pY}^{2}} = \frac{{E_{0\; X}i_{pX}^{2}} + {E_{0\; Y}i_{pY}^{2}}}{i_{p}^{2}}}$

In such a fashion, we are able to reduce an entire scan to a simplesubset of experimentally meaningful numbers.

CONCLUSION

We have devised an automated method of fitting that reduces a data scanto a small number of parameters from which all experimentally meaningfulinformation is extracted. Although we have focused on two-dimensional,vectoral data, simplified versions of the methods described here applyto 1-D scans. The important steps in the outlined procedure include: 1)assuming a model, 2) using vectoral sums to calculate an optimal phase,3) checking for behaviors that do not conform to the model, 4) making aninitial guess, using the inherent properties of the signal to minimizethe effects of aberrant noise, 5) iterate to optimize the fit, perhapswhile implementing a weighting scheme, 6) enforcing boundary conditions,7) detecting and correcting flawed fits, 8) judging fit suitability, and9) extracting meaningful quantitative information. By programming acomputer to follow this process, we have derived an automated methodthat extracts meaningful data from one scan in less time than it takesthe instrumentation to measure the next one.

Example 2 Rapid Extraction of Phase and Amplitude from a Noisy,Digitized Sine Wave of Known Frequency

In extremely clean systems that create pure sine waves, one candetermine a wave's phase by finding its zero-crossings and can measureits amplitude by finding local maxima and minima. If finding theseextrema is too difficult, one can instead calculate the RMS of the wave,in which case the amplitude will be the square root of 2) times theextracted RMS value. However, pure sine waves are rare in real worldsystems such as the systems described herein. Noise in the signal, makesfinding maxima, minima, and zero-crossings more difficult as methods toextract values. Furthermore, the RMS method measures power irrespectiveof frequency, so as the target signal decreases in amplitude, highfrequency noise begins to dominate and swamp out the true signal.

Non-linear regression techniques can reliably extract values from noisydata, but they are iterative processes, and as such can take largeamounts of computational time. This example uses only sums (anddifferences), and then renormalization and a basic coordinatetransformation. As such, it is extremely rapid. Also, the sums whichcomprise all of the data reduction (from some large number of points percycle to just two points per cycle) can easily be programmed into anembedded device, thus allowing more rapid data transfer from theacquisition device (e.g. the instrument of the invention) to the datamanipulation and storage device (e.g. computer).

The following equations are used to extract phase and amplitude from asinusoidal signal of known frequency that has been digitized such thatit has 4n points per cycle. The summations that create {tilde over (X)}and {tilde over (Y)} are a means of averaging away any random noise, andare simple enough to be programmed into the firmware.

If (in Cartesian coordinates) we plot

$y = {{R\; {\sin \left( {{2\pi \; {ft}} + \varphi} \right)}\mspace{14mu} {and}\mspace{14mu} X} = {R\; {\sin \left\lbrack {{2{\pi \left( {{ft} + \frac{1}{4}} \right)}} + \varphi} \right\rbrack}}}$

then we create a circle of amplitude R centered at the origin. If weplot only one quarter of a circle, then we are using the values fromhalf of one cycle (the first quarter for y and the second for x). Forthis subset, we can calculate average values of y and x, called y_(1/2cycle) and x _(1/2cycle).

${\overset{\_}{y}}_{U\; 2{cycle}} = {\frac{1}{{1/4} - 0}{\int_{n}^{1/4}{R\mspace{14mu} {\sin\left( {{{2\pi} + {\varphi_{i}{z}}} = {{\frac{2\; R}{\pi}\left( {{\sin \mspace{14mu} \varphi} + {\cos \; \varphi}} \right)} = {{\frac{{R \cdot 2}\sqrt{2}}{\pi}{\sin \left( {\varphi + \frac{\pi}{4}} \right)}\mspace{14mu} {and}{\overset{\_}{x}}_{V\; 2{cycle}}} = {\frac{1}{{1/2} - {1/4}}{\int_{1/4}^{1/2}{R\mspace{14mu} \sin {\quad\left( {{{2\pi} + {\varphi_{i}{z}}} = {{\frac{2\; R}{\pi}\left( {{\sin \mspace{14mu} \varphi} - {\cos \; \varphi}} \right)} = {\frac{{R2} \cdot \sqrt{2}}{\pi}{\cos \left( {\varphi + \frac{\pi}{4}} \right)}}}} \right.}}}}}}} \right.}}}}$

These values describe a point slightly within the arc of the quartercircle whose angle bisects the arc's sweep, and from them we can recoverthe original phase and amplitude:

$\varphi = {{{\arctan \left( \frac{\overset{\_}{y}}{\overset{\_}{x}} \right)} - {\frac{\pi}{4}\mspace{14mu} {and}\mspace{14mu} R}} = \frac{\sqrt{{\overset{\_}{x}}^{2} + {\overset{\_}{y}}^{2}}}{2\sqrt{2/\pi}}}$

Note that, to include information from the entire cycle when calculatingthese average values, we can simply use the fact that sin(x)=−sin(x+n/2)to find the same results:

$\overset{\_}{y} = {{\frac{1}{2}\left\lbrack {\frac{1}{{1/4} - 0}{\int_{0}^{1/4}{R\; {\sin \left( {{2\pi} + \varphi} \right)}\ {z}\frac{1}{{3/4} - {1/2}}{\int_{1/2}^{3/4}{R\; {\sin \left( {{2\pi} + \varphi} \right)}\ {z}}}}}} \right\rbrack} = {{\frac{{R \cdot 2}\sqrt{2}}{\pi}{\sin \left( {\varphi + \frac{\pi}{4}} \right)}\overset{\_}{x}} = {\frac{1}{2}\left\lbrack {{\frac{1}{{1/2} - {1/4}}{\int_{1/4}^{1/2}{R\; {\sin\left( {{{2\pi} + {\varphi \ {z}}} = {\frac{1}{1 - {3/4}}{\int_{1/4}^{1}{R\; {\sin \left( {{2\pi} + \varphi} \right)}\ {z}}}}} \right\rbrack}}}} = {\frac{{R \cdot 2}\sqrt{2}}{\pi}{\cos \left( {\varphi + \frac{\pi}{4}} \right)}}} \right.}}}$

We can exploit this behavior as follows: First, acquire an AC signal andlock into a desired frequency component. Choose the acquisition ratesuch that the number of points per cycle is evenly divisible by four—itcan be written as 4n where n is an integer. The first n pointscorrespond to the first quarter cycle, the second n points the secondquarter, etc.

If we add all n data points in the first quarter of the cycle andsubtract from them all n points in the third quarter, then we will havean unnormalized value, call it Y=2n= y, analogous to the y describedabove. Similarly, adding together the points in the second quarter andsubtracting those in the fourth will yield {tilde over (X)}=2n={tildeover (x)}. To extract the phase and amplitude from the data, we replacey and x by {tilde over (Y)}/2 and X/2n in the equations for (φ and Rabove, but with a few alterations necessitated by the digitization:

${\varphi = {{{\arctan \left( \frac{\overset{\_}{Y}}{\overset{\_}{X}} \right)} - {\frac{\pi}{4}\left( {1 + \frac{1}{n}} \right)\mspace{14mu} {and}\mspace{14mu} R}} = \frac{\sqrt{{\overset{\_}{X}}^{2} + {\overset{\_}{Y}}^{2}}}{2n\mspace{14mu}*\mspace{14mu} C_{n}}}},{where}$$C_{n} = {\frac{1}{n}\sqrt{\left( {\sum\limits_{i = 1}^{n}\; {\cos \left( {\frac{\pi}{2} - \frac{i}{n}} \right)}} \right\rbrack^{2} + \left\lbrack {\sum\limits_{i = 1}^{n}\; {\sin \left( {\frac{\pi}{2} - \frac{i}{n}} \right)}} \right\rbrack^{2}}}$

In the above equation for R, C_(n) has the following values:

n C_(n) n C_(n)  1 1.000000 12 0.900959  2 0.923880 13 0.900864  30.910684 14 0.900789  4 0.906127 15 0.900728  5 0.904029 20 0.900548  60.902893 25 0.900464  7 0.902208 30 0.900419  8 0.901764 50 0.900353  90.901460 70 0.900335 10 0.901243 90 0.900328 11 0.901082 inf 0.900316(As n becomes very large, these equations approach those described abovefor the continuous case. 1/n goes to zero, and the value of 0.900316listed for C_(inf) is actually two times the square root of two overpi.)

The equations for φ and C_(n) the previous page describe a procedure bywhich we can extract phase and amplitude from a sinusoidal signal ofknown frequency that has been digitized such that it has 4n points percycle. The summations that create {tilde over (X)} and {tilde over (Y)}are a means of averaging away any random noise, and are simple enoughthat they can be programmed into the firmware.

Example 3

DNA hybridization assays on an eSensor™ chip typically require anincubation time of 4 to 6 hours for a 10 nM range target concentration.This time period was chosen based on diffusion based hybridization timesin large volume (i.e., 0.5 ml) cartridges. However, for smaller volumecartridges (i.e., 100 μl), a 4 to 6 hour incubation time is notsufficient to achieve saturated hybridization signals. Thus, a number ofdifferent convective mixing techniques were evaluated in an eSensor™chip in order to accelerate DNA hybridization.

Experimental Protocols and Materials:

HFE-H was chosen as the model assay system. All chips and reagents usedwere from the same stock in order to eliminate sample or chipperformance variations:

-   -   Chips used: DC_(—)668    -   Capture probe: D2002    -   Target concentration: 10 nM (unless otherwise specified)    -   Signaling probes: D2005 and D2004 (300 nM each)    -   Hybridization buffer: prepared according to standard protocols    -   Heating systems: Depending on the type of mixing used, different        methods of heating were employed.    -   For example, chips were incubated in a convective oven or on        chip heating devices were used.    -   Controls: Diffusion based hybridization was used as the control

Horizontal vs Vertical Orientation of Chip:

Method of heating chips: convective oven at 35° C.

Slow hybridization kinetics was observed in chips that were incubated ina horizontal orientation for 100 μl volume cartridges. At 10 nM targetconcentration chips incubated horizontally did not reach saturationsignal values by 5 hours. However, if the concentration of the targetwas increased to 100 nM, saturation signal values were observed within 5to 6 hours.

Increasing the thickness of the chamber also improved the performance ofthe horizontally incubated chip due to increased volume/z-dimension. SeeFIG. 70A.

Chips that were incubated vertically performed better and reachedsaturation signal values with 2 to 3 hours for a 10 nM targetconcentration.

Recirculation Pumping

Method of Heating: convective oven at 35° C.

Chips were attached to a mini-peristaltic pump using micro bore peektubing with a total dead volume of nearly 7 μl (<10% of chip volume of8- to 100 μl). The fluid was recirculated at approximately 100 μl/min.The entire pump, tubing and chip assembly was placed inside an oven at35° C. Measurements were made in real time by placing theeSensor™-600board inside the oven. Saturation signals for the pumpedchips were obtained within 1 hour. See FIG. 70B.

Bubble Assisted Piezo (PZT) Mixing

Cartridge covers were drilled with 500 μm diameter holes that served assites for bubbles when the hybridization buffer was added to the chip. Apiezo-electric transducer was glued on the back of the cartridge andexcited with 5 Khz a.c. waveform at 10Vp-p. This device was operated intwo modes, a square wave or a sine wave excitation waveform. PZT basedchips performed better than diffusion controls, requiring 2.5 hours toreach saturation. See FIG. 70C.

Thermal Gradient Based Mixing

A thermal gradient was created across an assembled chip by heating thebottom of the chip to 65° C. and cooling the top of the cartridge coverto 10° C. The thermal gradient was created by jacketing the chip betweentwo peltier heaters. The diffusion control chip was heated using apeltier heater rather than the convective oven. Using peltier beatingimproved hybridization kinetics in the vertical diffusion control chipsuch that a saturation signal was observed within 2 hours. The chip inwhich thermal gradient mixing was used performed even better and reachedsaturation values within one hour. See FIG. 70D.

Biochannel with Bubble Pump Based Mixing

A microchannel was place on top of the eSensor™ chip by cutting adhesivetape into approximately 1 mm wide thin strips and placing these stripson top of the eSensor™ chip. A bubble was intentionally introduced intoone corner of the chip. This bubble was then utilized to enhance mixingby alternately expanding and contracting the bubble volume by heatingand cooling the bubble area. This approach allows mixing due to thepressure flow created by changing the volume of the bubble within thechip. However, the addition of microchannels decreased the chip volumeto approximately 20 μl, resulting in slower kinetics in this system.Using the bubble pump accelerated the kinetics, but the kinetics werestill not as good as observed with other mixing techniques. Typicalresults are shown in FIG. 70E.

Acoustic Streaming

The proprietary technology of Covaris, Inc. was used to generate mixingin the chip. In this method, a fluid jet surrounds the chip cartridge,through which acoustic waves are transferred to the chip. A non-standardcartridge of 20 μl was used and the assays were performed withouttemperature control as no temperature control is currently feasible withthis system. Saturation signal levels were lower using this systemregardless of whether mixing was used. Typical results are shown in FIG.70F.

CONCLUSIONS

Saturation Time to Signal Saturation Rate Enhancement Over MixingTechnique (nA) (hours) Diffusion Control Vertical Diffusion 50 3 notapplicable (35° C., oven) Vertical Diffusion 60 2 not applicable (35°C., peltier) Thermal Gradient 60 1   2X Recirculation Pumping 50 1   3XBubble Assisted 50 2.5 1.2X Acoustic Streaming

1-14. (canceled)
 15. A multiplex detection device, comprising: aplurality of stations each for housing a biochip and each independentlyaddressable; a plurality of biochips each in a station and eachcomprising: i) an array of working electrodes at least some of whichcomprise a capture binding ligand for detecting analyte in an aqueoussample; ii) one or more reference electrodes; iii) one or more auxiliaryelectrodes; and iv) interconnects for electrical communication with eachof said electrodes; controls for inputting signal to said electrodes; adetector for detecting output signal from said electrodes; one or morepumps for circulating aqueous analyte samples on said biochips; and oneor more processors for amplifying analyte signal or filteringbackground.
 16. The device of claim 15 further comprising one or more ofan analog to digital converter and a digital to analog converter. 17.The device of claim 15 wherein at least some of said working electrodescomprise a self assembling monolayer.
 18. The device of claim 17 whereinsaid self assembling monolayer is a mixed self-assembling monolayercomprising insulators.
 19. The device of claim 15 comprising differentcapture binding ligands on different working electrodes.
 20. The deviceof claim 15 wherein said biochips further comprise one or moreelectrochemical detection labels.
 21. The device of claim 20 comprisinga plurality of electrochemical detection labels of differentelectrochemical potential.
 22. The device of claim 21 wherein at leastsome of said electrochemical detection labels of differentelectrochemical potential are selected from the group consisting offerrocene and ferrocene derivatives.
 23. The device of claim 15 whereinat, least one of said capture binding ligands comprises nucleic acids ornucleic acid analogs.
 24. The device of claim 15 further comprising aplurality of different electrochemical detection labels of differentelectrochemical potential on each biochip.
 25. The device of claim 15comprising a plurality of different nucleic acid capture binding ligandson each biochip.
 26. The device of claim 15 comprising both nucleicacids and non nucleic acid capture binding ligands.
 27. The device ofclaim 15 wherein said biochips further comprise nucleic acid analyteamplification reagents.
 28. A multiplex detection device, comprising: aplurality of stations each for housing a biochip and each independentlyaddressable; a plurality of biochips each in a station and eachcomprising: i) an array of working electrodes at least some of whichcomprise a capture binding ligand for detecting analyte in an aqueoussample; ii) one or more reference electrodes; iii) one or more auxiliaryelectrodes; iv) interconnects for electrical communication with each ofsaid electrodes; and v) one or more aqueous buffer or reaction chambers;controls for inputting signal to said electrodes; a detector fordetecting output signal from said electrodes; one or more pumps forcirculating aqueous analyte samples on said biochips; and one or moreprocessors for amplifying analyte signal or filtering background; one ormore on-chip aqueous buffer or reaction mixture chambers.
 29. Amultiplex detection device, comprising: a plurality of stations each forhousing a biochip and each independently addressable; a plurality ofbiochips each in a station and each comprising: i) an array of workingelectrodes at least some of which comprise a capture binding ligand fordetecting analyte in an aqueous sample; ii) one or more referenceelectrodes; iii) one or more auxiliary electrodes; iv) interconnects forelectrical communication with each of said electrodes; and v) one ormore aqueous buffer or reaction chambers; wherein said plurality ofbiochips are configured to circulate and recirculate an aqueous analytesample across said array of working electrodes; controls for inputtingsignal to said electrodes; a detector for detecting output signal fromsaid electrodes; one or more pumps for circulating aqueous analytesamples on said biochips; and one or more processors for amplifyinganalyte signal or filtering background; one or more on-chip aqueousbuffer or reaction mixture chambers.
 30. A multiplex detection device,comprising: a plurality of stations each for housing a biochip and eachindependently addressable; a plurality of biochips each in a station andeach comprising: i) an array of working electrodes housed serially in anelongated channel and at least some of which comprise a capture bindingligand for detecting analyte in an aqueous sample; ii) one or morereference electrodes; iii) one or more auxiliary electrodes; iv)interconnects for electrical communication with each of said electrodes;and v) one or more aqueous buffer or reaction chambers; wherein saidplurality of biochips are configured to circulate and recirculate anaqueous analyte sample across said array of working electrodes; controlsfor inputting signal to said electrodes; a detector for detecting outputsignal from said electrodes; one or more pumps for circulating aqueousanalyte samples on said biochips; and one or more processors foramplifying analyte signal or filtering background;
 31. A multiplexdetection device, comprising: a plurality of stations each for housing abiochip and each independently addressable; a plurality of biochips eachin a station and each comprising: i) an array of working electrodes atleast some of which comprise a capture binding ligand for detectinganalyte in an aqueous sample; ii) one or more reference electrodes; iii)one or more auxiliary electrodes; and iv) interconnects for electricalcommunication with each of said electrodes; controls for inputtingsignal to said electrodes; a detector for detecting output signal fromsaid electrodes; one or more pumps for circulating aqueous analytesamples on said biochips; one or more processors for amplifying analytesignal or filtering background; and thermocontrols for controlling thetemperature of said biochips.
 32. The device of claim 15 wherein atleast one of said analytes or capture binding ligands is associated witha genetic or infectious disease.
 33. The device of claim 23 wherein saidnucleic acid analog has a positively charged backbone.
 34. The device ofclaim 15 wherein said biochips further comprise nucleic acid signalprobes bearing electrochemical detection labels.
 35. The device of claim15 wherein each of said plurality of biochips further comprises a memorychip or barcode and wherein said device further comprises a memory chipor barcode reader.
 36. The device of claim 15 wherein said plurality ofbiochips are configured to receive and process an aqueous sampleselected from the group consisting of blood, tissue, sputum and saliva.37. The device of claim 15 wherein said analyte or analytes is presentin the form of an aqueous sample and wherein said device furthercomprises mixing means selected from the group consisting ofpiezoelectric mixing, thermal gradients, bubble pump mixer, and acousticstreaming to facilitate distribution of said analyte or analytes to saidworking electrodes.
 38. The device of claim 15 further comprisingdigital lock-in means and a program for performing one or more of aFourier transform and joint time-frequency transformation to analyzedetection results.
 39. The device of claim 15 further comprising meansfor measuring one or more of a frequency, amplitude, phase shift, DCoffset voltage, and faradaic impedance response.